#gradient centrifugation protocol

[uberstrainer]

Why Labs Are Choosing PluriMate: Key Benefits of Non-Disruptive Cell Separation

Laboratories aiming to separate leukocytes and peripheral blood mononuclear cells (PBMCs) with minimal contamination are increasingly turning to tools that leverage the density gradient centrifugation principle. Among these, PluriMate stands out due to its innovative design that simplifies cell separation while maintaining sample purity. PluriMate's unique sponge barrier enhances efficiency, making it a preferred choice for labs conducting regular cell isolation from whole blood or bone marrow.

1. Simplified Sample Preparation Process

One of the key benefits of PluriMate is its ease of use. Traditional cell separation methods often involve careful and time-consuming layering of blood over the separation medium, which requires skill and can increase the risk of cross-contamination. PluriMate eliminates this need entirely. Researchers can pour anticoagulated blood or bone marrow directly into the PluriMate tube without worrying about manual layering. This not only saves valuable time but also reduces the potential for errors, making it an ideal tool for labs looking to streamline their processes.

2. Innovative Particle Separation Techniques

PluriMate utilizes a built-in porous sponge made from high-grade polyurethane that acts as a non-disruptive barrier, facilitating reliable particle separation techniques. During centrifugation, this barrier prevents unwanted cells—like erythrocytes and granulocytes—from mixing with the desired cell population. The density gradient within the PluriMate tube separates leucocytes, lymphocytes, and PBMCs based on their specific densities, allowing them to collect in an interphase above the separation medium. This layer of targeted cells can then be easily harvested without contamination from denser, undesired cells. PluriMate’s particle separation techniques ensure that researchers receive high-quality samples for downstream applications.

3. Reduced Risk of Contamination

Contamination is a significant concern during cell separation, especially in multi-step processes where each step increases exposure to contaminants. PluriMate addresses this with its non-disruptive design that minimizes the need for handling. Once centrifugation is complete, the sponge barrier remains intact, preventing recontamination of the enriched cell fraction as it is harvested. This design is particularly beneficial when processing large batches or working with valuable samples, as it ensures that the isolated cells remain pure and ready for analysis or further applications, such as cell enrichment studies or cell-based assays.

4. Efficient Particle Filtration

Effective particle filtration is essential for isolating high-quality cell populations. PluriMate’s porous sponge acts as a built-in filter, preventing the mixture of the sample material with the separation medium, thereby preserving the integrity of the cells that need to be isolated. By keeping unwanted particles and cells out of the desired layer, PluriMate provides a reliable way to filter and enrich the sample without additional equipment. This feature makes it particularly attractive for labs seeking straightforward solutions for cell separation and filtration, with minimal need for extra tools or manual intervention.

5. Versatile for Various Applications

PluriMate is adaptable to both large and small sample volumes, making it a versatile tool in laboratories with diverse sample processing needs. Whether pre-filled with the separation medium or provided as an unfilled tube, PluriMate offers flexibility for a wide range of cell separation protocols. Additionally, it can be combined with pluriSpin for negative cell separation, further expanding its utility. This versatility allows researchers to conduct multiple applications, including cell enrichment and density-based separations, making it an invaluable addition to modern labs.

Conclusion

For labs prioritizing efficiency, reduced contamination, and reliable separation, PluriMate is an effective solution that operates on the density gradient centrifugation principle. With its innovative sponge barrier, easy handling, and adaptability to various cell separation tasks, PluriMate meets the demands of researchers focused on high-quality results. By simplifying the cell separation process, PluriMate enables laboratories to achieve consistent outcomes while maintaining sample purity, making it a top choice for professionals in the field of particle separation and cell isolation.

[realdrake]

Purifying nanoparticles into monodisperse fractions is a crucial step in understanding their physical properties and minimizing variability in biological applications. The lack of uniformity in nanoparticle samples can lead to inconsistent results, making it challenging to draw meaningful conclusions. While various methods have been explored for purifying "hard" nanoparticles, few studies have focused on "soft" nanoparticles like polymersomes.

Polymersomes, with their unique physico-chemical and mechanical properties, are attractive for biological and medical applications. However, their self-assembly process inherently leads to broad size distributions and high variability in morphology. This heterogeneity can result in a mixture of spherical vesicles, tubes, and genus structures, as well as non-vesicular structures like micelles. To address this challenge, we compared four complementary techniques for separating polymersomes by size and shape: cross-flow filtration (CFF), differential centrifugation (DC), size exclusion chromatography (SEC), and density gradient centrifugation (DGC).

the  results show that each technique has its advantages and disadvantages. CFF efficiently separates micelles from polymersomes, possibly due to a combination of size exclusion and differential fluid dynamics. However, it cannot be used to separate sub-populations of polymersomes by size. DC and SEC enable separation of polymersomes into distinct size fractions, but result in sample concentration loss. DGC, on the other hand, achieves shape-based separation by exploiting differences in membrane packing density.

they found that the different shape of polymersomes corresponds to changes in the density of membrane packing, providing the means for their separation by the DGC-based method. This approach allows for the separation of polymersomes into distinct fractions based on their shape, which is essential for understanding their physical properties and biological behavior.

By combining these techniques, we can develop efficient purification protocols for polymersomes and other nanoparticles. This study demonstrates the importance of purification in producing consistent and reproducible results in biological experiments and improving application development in medicine and drug delivery. The ability to purify nanoparticles into monodisperse fractions will enable researchers to better understand their physical properties and behavior, ultimately leading to the development of more effective nanoparticle-based therapies.

[theblogs2024]

Maximizing Precision In Biomedical Investigation: Exploring Axis-Shield Density Gradient Media

While in the realm of biomedical analysis, The hunt for precision is unyielding. Researchers regularly search for advanced applications and procedures to isolate, purify, and analyze Organic parts with unparalleled accuracy. Axis-Protect Density Gradient Media emerges being a cornerstone Remedy, giving a transformative approach to separation and purification throughout various purposes.

Axis-Defend Density Gradient Media operates over the theory of density gradient centrifugation, a method that exploits the different buoyant densities of particles to realize meticulous separation. By layering samples atop meticulously calibrated gradient media and subjecting them to centrifugal forces, distinct bands or levels enriched with particular components emerge, facilitating specific isolation and analysis.

The flexibility of Axis-Shield Density Gradient Media can be a testomony to its efficacy. No matter whether isolating cells, organelles, or particles from complex biological mixtures, its tailor-made formulations cater into a spectrum of exploration needs. In addition, its compatibility with several centrifugation protocols and devices ensures seamless integration into laboratory workflows, empowering researchers with unparalleled flexibility.

In medical diagnostics, Axis-Shield Density Gradient Media performs a pivotal position in processes for example blood fractionation. By facilitating the separation of blood components such as erythrocytes, leukocytes, and platelets, it makes certain the Secure and productive provision of blood merchandise when reducing the potential risk of adverse reactions in patients.

The purity and reproducibility of Axis-Shield Density Gradient Media are exemplary. Researchers can rely on steady effects even in essentially the most demanding experimental situations, making it an indispensable tool in fields where precision is paramount, such as drug improvement, biomanufacturing, and past.

Moreover, Axis-Shield Density Gradient Media accelerates discoveries in elementary research. By enabling the isolation of specific mobile populations or subcellular organelles, it elucidates the intricacies of biological units, giving insights into immune responses, cellular signaling pathways, and disease mechanisms.

As biomedical exploration improvements, the demand for exact separation strategies proceeds to expand. Axis-Protect Density Gradient Media stands being a beacon of innovation, empowering researchers to examine the complexities of existence within the molecular degree. Its role being a catalyst for discovery and advancement underscores its significance in shaping the way forward for biomedicine and past.

Learn more info. check out here: axis shield

[molsons112000]

So you take a sample of lumpatic fluid at the same time. You take a sample of the blood.

The lymphatic is the waste system and you can compare and contrast that to what's in the blood. So you can see what's coming into the body. Say eating food and medicine. And what's in the circulatory system? And then what's going into the waste system the system?

This is what pharmaceutical firms never test and see how much of their drugs end up in the lymphatic system compared to in the blood and then testing the cells through a biopsy.

Even with brain matter they can get a single cell sample or multiple cell samples using this needle system.

A needle biopsy is a medical test that can help identify the cause of an abnormal lump or mass in the body. During a needle biopsy, a doctor guides a thin, hollow needle through the skin and into the area of interest. A 

syringe

 attached to the needle suctions out a small 

sample

 of cells and sometimes fluid. The doctor may numb the biopsy site, and the process may be repeated several times until enough cells are collected. 



Mayo Clinic

Needle biopsy - Mayo Clinic

Aug 20, 2022 — During the needle biopsy, the doctor guides a needle through your skin and into the area of interest. A sample of cells is collected and the needle is withdrawn. This process may be repeated several times until enough cells are collected. Common types of needle biopsy techniques include:



Cancer.Net

Fine Needle Aspiration Biopsy: How to Prepare and What to Expect | Cancer.Net

Oct 7, 2021



uofmhealth.org

Needle Biopsy | University of Michigan Health

A needle biopsy is a medical test which can identify the cause of an abnormal lump or mass in your body. A radiologist performs this procedure in the radiology department. During the procedure, the radiologist inserts a small needle into the abnormal area and the sample is then sent to the pathologist for analysis.

A needle biopsy is less invasive than open and closed surgical 

biopsies

. It's typically an 

outpatient procedure

 with very infrequent complications, and in over 90 percent of patients, needle biopsy provides enough tissue for the pathologist to arrive at a diagnosis. 

The recovery time is usually quick, though there might be some bleeding and/or 

bruising

. Core needle biopsies usually result in more bruising than a breast 

fine needle biopsy

. You may experience tenderness and 

soreness

 in the biopsy site for 1-2 days. You can take Tylenol or other pain relievers to relieve any discomfort or pain. 

They need to get this process down on mouse's first and then they can take it to humans.They get cell samples from the brain a cell biopsy of the brain.

Here are some steps for preparing a single-cell suspension from mouse brain tissue:

Rinse the tissue with sample preparation medium

Transfer the brain tissue into a strainer

Use the rubber end of a syringe plunger to push the digested brain tissue through the strainer

Rinse the strainer with more sample preparation medium

Centrifuge the suspension at 300 x g for 10 minutes with the break set to low

If the filter becomes clogged, transfer the contents to a new pre-wetted filter 





STEMCELL Technologies  ·  

YouTube · 2y

How to Prepare a Single-Cell Suspension from Mouse Brain Tissue

if the filter becomes clogged transfer the contents to a new pre-wetted. filter centrifuge at 300 times g for 10 minutes with the break set to low.



STEMCELL Technologies

How to Prepare a Single-Cell Suspension from Mouse Brain Tissue Protocol

The basic steps for preparing a single-cell suspension include:

Increasing the surface area of the solid tissue material

Digesting the extracellular matrix

Cleaving cell-cell junctions 

Other steps include:

Enzymatic digestion and mechanical trituration of 

vibratome



 sections

Removal of cellular debris with density gradient 

centrifugation



Collection of single cells by FACS

Library generation and quality control

Sequencing of single-cell transcriptomes using Illumina 

Microinjection



 into single cells in brain tissue is a technique to study and manipulate 

neural



 stem cells. However, it requires expertise and is a low-throughput process. 



National Institutes of Health (NIH) (.gov)

https://www.ncbi.nlm.nih.gov › pmc

Obtaining Acute Brain Slices - PMC

by T Papouin · 2018 · Cited by 32 — Procedure · Quickly extract the brain. Pour about half (150 ml) of the ice-cold slicing ACSF into the plastic

So now you can take a sample of the lymphatic fluid a sample of the blood and a sample of different cells in the body to compare this for the health of the individual. And how nutrition and pharmaceuticals are entering these cells.....

This will help all forms of medical diagnosis and pharmaceutical companies and doctors and hospitals & general...

This means misdiagnosis will almost be eliminated and you can detect if there's any cancer cells in the Lynthetic fluid and look for It in the blood. Then you could do a single cell sample of that tissue in that area. You think like the breast to see if there's any cancer? So this sample can be sent in and DNA matched to the patient. And you can carbon date the sample so they can't fake the diagnosis using old samples.

How they can genetically.Match the tissue sample and the lymphatic sample or any fluid sample to the patient. They can also date when that sample was created. Making sure no one's using old dna Information to cause insurance fraud.



Nature

www.nature.com

A combined method for DNA analysis and radiocarbon dating ...

by P Korlević · 2018 · Cited by 49 — Here we present a method that makes it possible to obtain both ancient DNA sequences and radiocarbon dates from the same sample material

Wikipedia

https://en.m.wikipedia.org › wiki

Radiocarbon dating

Radiocarbon dating is a method for determining the age of an object containing organic material by using the properties of radiocarbon, a radioactive ...

Missing: dna ‎| Show results with: dna



University of Chicago News

https://news.uchicago.edu › explainer

Carbon-14 dating, explained - UChicago News

Radiocarbon dating works on organic materials up to about 60,000 years of age. Conventional radiocarbon dating requires samples of 10 to 100

So any genetic material from a crime scene and they should be testing the lymphatic system at the corner's office. It is another place to look for poison. It might be out of the blood system, but still in the lymphatic system. So it's another way to check if somebody is getting poisoned.....

So they should be testing mummies for poisoning and you can test bone Merrill.As well.

There is another way for the corner and while the person's alive to test for poisoning is take a sample of the bone marrow. But it's also another way to check their health.

National Institutes of Health (NIH) (.gov)

https://www.ncbi.nlm.nih.gov › pmc

Dynamic observation of bone marrow suppression and ...

by Y Liu · 2022 · Cited by 1 — In one of our patient, 1 ng/mL of colchicine was detected in the bone marrow sample at 10 d after poisoning (poison

So watching Jimmy Kimmel as I put this together.

[predictoreports]

The global market size of Cesium chloride is $XX million in 2018 with XX CAGR from 2014 to 2018, and it is expected to reach $XX million by the end of 2024 with a CAGR of XX% from 2019 to 2024. There are 3 key segments covered in this report: geography segment, end use/application segment, and competitor segment. For geography segment, regional supply, application-wise, and type-wise demand, major players, price is presented from 2013 to 2023. This report covers the following regions: * North America * South America * Asia & Pacific * Europe * MEA (the Middle East and Africa) The key countries in each region are taken into consideration as well, such as United States, China, Japan, India, Korea, ASEAN, Germany, France, UK, Italy, Spain, CIS, and Brazil, etc. For end use/application segment, this report focuses on the status and outlook for key applications. End users sre also listed. * Application I * Application II * Application III For competitor segment, the report includes global key players of Cesium chloride as well as some small players. The information for each competitor includes:

[uberstrainerusa]

Protocol For Buffy Coat Preparation From Whole Blood

In this blog, we’ll explore what is buffy coats and how it is prepared from whole blood. We will also learn how Pluribead and Plurispin help in this process.

Millions of cells are suspended in a liquid called plasma to form blood. Red blood cells (which carry oxygen), white blood cells (which fight infection), and platelets are examples of these cells (which help with clotting). Examining red blood cells is simple due to their abundance; thousands of red blood cells can be seen in a single drop of blood. White blood cells, on the other hand, are present in very small numbers and are more difficult to examine.

Examining a buffy coat smear is the quickest way to examine large numbers of white blood cells. The buffy coat is simply a collection of all of the white blood cells and platelets in a blood sample.

Let’s find out how Pluribead Antibody Cell Separation technology helps in buffy coat extraction.

Preparation Of Buffy Coat From Scratch

Making your own buffy coat is an alternative. Therefore, simply adhere to the following succinct protocol:

Mix one part washing buffer with one part whole blood.

Centrifuge the diluted whole blood for 10 minutes at 200 x g while the brake is turned off.

Remove the interphase leukocytes (buffy coat)

It's important to remember that a whole blood sample could contain pathogens, so these samples must be handled with the same care as if they were capable of transmitting infectious diseases. The time has come to separate the buffy coat from a whole blood sample after all the necessary preparations have been made.

Buffy Coat Extraction

The buffy coat is separated from the plasma and RBC by centrifuging the prepared whole blood sample. After centrifugation, there will be a thin layer between the RBC and plasma that accounts for about 1% of the sorted sample – the buffy coat. The experimenter uses a tiny pipette to gather and transfer the buffy coat to a different container.

The buffy coat is in direct contact with isolated RBCs, while PBMCs are isolated from the other particles in the sorted sample by density gradient centrifugation. Using a manual extraction technique like pipetting increases the possibility that contaminating RBCs will be present in the buffy coat sample because of the size and abundance of RBCs (Buffy coats are referred to as "pink" before further processing because of this). Researchers frequently combine centrifugation with another cell separation technique, like BACS, to get rid of any remaining RBC contamination in order to enrich the buffy coat even more.

How can Plurispin be useful?

A brand-new technique for isolating negative cells from whole blood, buffy coat, or cord blood is called the PluriSpin system. This new technique isolates live, unaltered, and highly purified cells in a single step without the use of magnets or a column. As a result, there is less chance of activating or harming the target cells.

How PluriBead can help?

PluriBead is a one-of-a-kind Antibody Cell Separation technology that doesn't rely on magnetic components. The process is simple: Your pluriBeads are sieved through a strainer with your bound target cells on top, while the unwanted cells pass through. After detaching, you have your target cells ready.

For more information, you can visit our website. Experience the difference for yourself by using our products. Get started with our cell separation products today!

[digitalsomnath]

Fast and gentle isolation of untouched platelets using PluriSpin

Searching for an efficient tool to isolate the pure, viable, and untouched platelets-low population from whole blood? Read on to learn about PluriSpin Human PLT Depletion.

PluriSpin Human PLT Depletion is a negative cell separation method used to isolate pure, viable, and untouched platelets-low populations from whole blood, buffy coat, cord blood, or similar sample material. The Platelet is labeled by the product, while the isolated human platelets-low population is left in the sample.

Solution and advantages with pluriSpin Human PLT Depletion

 ·         Without the use of beads, depletion occurs.

·         Increases the purity of enriched cells in a single step by using density gradient centrifugation.

·         Can be used in conjunction with negative separation techniques such as pluriSpin.

·         The enrichment of rare cells will be improved (e.g. in use with pluriSpin CD45 Depletion)

·         Because of the shorter centrifugation step, the blood sample is subjected to the least amount of manipulation (compared to standard protocol with platelet density gradient)

·         The enrichment of cells from blood samples older than 8 hours will be enhanced.

 Human PLT Depletion Protocol

 Required Materials

• Mixing device w/ rotation and tilting, e.g. pluriPlix (50-01010-80)

• Wash Buffer, e.g. 10x Wash Buffer 500 ml (60-00080-11)

• Density gradient medium

 Label

1. Restart the PluriSpin suspension (i.e. vortex 20 sec).

2. Add PluriSpin suspension at a rate of 20 l/ml of whole blood (e.g., 80 l of suspension for 4 ml of whole blood). Combine thoroughly (i.e vortex 2 sec).

 Incubate

3. Incubate at room temperature for 15 minutes on a PluriPlix or rolling mixer with tilting and 15 rpm rotation.

4. Gently mix the sample with an equal volume of wash buffer.

5. Spread the diluted sample over the density gradient medium.

 Spin

6. Centrifuge at 800 x g for 15 minutes at room temperature with the brake off.

 Collect

7. Remove the enriched cells with care from the density gradient medium: plasma interface.

8. After collecting the cells from the interface, vortex for 5 seconds to break up the aggregation.

 Wash

9. Fill the reaction tube halfway with wash buffer.

10. Spin down cells for 10 minutes at 4°C with 300 x g (no or small brake).

11. Remove the supernatant and set the reaction tube on the table for 20 seconds. Excess wash buffer will run down the tube wall and collect at the bottom.

12. Remove the majority of the liquid above the pellet. (The liquid will appear foggy because these are mostly platelets – aspiration will improve washing results)

13. Carefully pipette the pellet with 1 ml of wash buffer.

14 Repetition of steps 9–12.

15. Reconstitute the pellet to the desired volume.

 As needed, use enriched cells. If red blood cells (RBC) are present, they can be lysed with Ammonium Chloride Solution prior to flow cytometric analysis. We recommend that you always use CD45 staining to distinguish white blood cells from debris, platelets, and RBC.

[juniperpublishers-gjorm]

Optimization of Sperm Culture Conditions for Human Assisted Reproductive Technologies

Authored by:  Charles L Bormann*

Optimizing sperm performance can be a differentiating factor in clinical pregnancy rates for assisted reproductive technology (ART) laboratories. Minimizing cellular stress during routine sperm processing by maintaining a precise and stable environment is of great importance in the field. In commercial media, pH of the external cellular culture environment (pHe) is typically maintained at 7.2-7.4 by various buffering reagents, while the internal pH of the cell (pHi) depends on available lactate and amino acids. Combining buffers in solution has been previously shown to be valuable for stabilizing internal and external pH for various biological systems. We hypothesized that a dual-buffer culture medium might improve sperm performance for ART. Here we demonstrate superior performance of a commercially available dual-buffer solution of HEPES and MOPS: Multipurpose Handling Medium (MHM, Irvine Scientific). Significantly better performance, assessed at 8 and 48 hours, and measured as: sperm viability, total motility, and rapid forward progression, was observed for MHM over single buffer controls.

Keywords: Sperm processing; Sperm wash; Sperm viability; Multipurpose handling medium

Abbreviations: ART: Assisted Reproductive Technology; MHM: Multipurpose Handling Medium; SPWASH: Sperm Washing Medium; CASA: Computer Assisted Sperm Analyzer; IQR: Inter Quartile Range; MHM: Multipurpose Handling Media; AAB: American Association of Bioanalysts

Introduction

Achieving and maintaining high pregnancy rates is a top priority for embryology labs and fertility practices. Optimizing sperm performance can be a key differentiating factor in achieving better clinical pregnancy rates. Human sperm handling procedures for ART are commonly performed under atmospheric conditions, where temperature and CO2, play a significant role in regulating pH. Maintaining a precise and stable pH balance is challenging, as even minor environmental fluctuations can negatively impair human sperm function. HEPES and MOPS are zwitter ionic organic buffers with pKa at 20 °C of ~7.55 and 7.15, respectively. Individually, they have been extensively studied in numerous ART-related processes, including: sperm isolation, oocyte retrieval, ICSI, embryo biopsy, embryo transfer, and cryopreservation. However, recent evidence supports the notion that combining HEPES: MOPS may allow for improved media formulations.

Multipurpose Handling Medium (MHM, Irvine Scientific) is a commercially available dual-buffer solution of HEPES and MOPS used to maintain stable conditions for oocytes, and embryos when being manipulated under atmospheric conditions. Dualbuffer solutions have demonstrated improvements in working pHe, Na+ or K+ concentration, and/or concentration toxicity and osmolality, over single buffer media, and is specially formulated to maintain a physiological pH of 7.2-7.4 over a broad temperature range. We hypothesize that a dual-buffer Multipurpose Handling Medium will better support and maintain sperm viability parameters compared to specimens processed with a single buffer solution.

Here we describe the baseline conditions for human semen samples in three handling media (dual buffer MHM compared to two commercially available single buffer HEPES media: a control sperm washing medium, Sperm Washing Medium (SPWASH, Irvine Scientific), and an ART handling medium, Quinn's Advantage Medium with HEPES (QUINN, Cooper Surgical), by distribution of motility percent and rapid progression percent, and we quantify causal association of motility percent and rapid progression percent to handling medium after 48 hours at room- atmosphere conditions.

Materials and Methods

Sperm collection, preparation and processing

Semen samples from 21 men undergoing routine fertility testing and having normal specimen parameters as defined by the WHO 5thedition [1] were included in this study. Specimens were liquefied for 30-60 minutes and sperm were quantified using a Computer Assisted Sperm Analyzer (CASA; CEROS™ II, Hamilton Thorne). Specimens meeting eligibility criteria were layered over a 50:90% Isolate gradient (Isolate, Irvine Scientific) and centrifuged for 20 minutes at 300xg. Following centrifugation, sperm pellets were suspended with 10ml of SPWASH and centrifuged for 10 minutes at 300xg. This wash step was performed two times for each specimen. The final sperm pellet was quantified using CASA and equivalent concentrations of motile sperm (~5million/ml) were added to the following treatment groups: SPWASH, QUINN, and MHM. All specimens were analyzed under IRB approval.

Data collection and statistics

Following collection, processing, and preparation of participants semen samples in three handling media- labeled and capped tubes were left in a controlled room temperature environment for 48 hours. Assessments were performed at 8, 24, and 48 hours, at which time the concentration, motility percent, rapid motile progression percent, medium motile progression percent, slow motile progression percent, and static motile percent of total sperm were recorded. The mean and standard deviation of the recorded baseline (0-hour) characteristics were calculated to describe the observed location and spread of these variables, by handling medium and overall. The distributions of motility percent and rapid progression percent within each handling medium are summarized in box plots at baseline and each follow-up time.

The overall association between motility percent and handling medium was assessed using a linear mixed effects regression model. This model estimates the average motility percent of sperm in each medium, adjusting for the motility percent at baseline and accounting for possible correlation between measurements on the three samples taken from the same individual. Separate models were used to estimate motility percent at 8 hours and at 48 hours. Analogous linear mixed effects models were fit to assess the association between rapid progression percent and handling medium at 8 hours and at 48 hours.

Results

Semen samples from 21 individuals, and each sample was prepared following the same laboratory protocol and divided into three subsamples, one in each of the study media (MHM, QUINN, SPWASH), for a total of 63 samples. The study design is balanced across subjects and sperm handling media, and there is no missing data for the analyzed variables. The mean and standard deviation of baseline characteristics of the samples are shown in Table 1. The concentration of sperm is similar across handling media, and there were no statistical differences in total motility or motility progression categories across the three treatment groups. Box plots comparing the distributions of motility percent in MHM, QUINN, and SPWASH at baseline and at 8, 24, and 48 hours are shown in Figure 1. In these plots, the horizontal black line dividing the box indicates the median (or 50th percentile) of motility percent for samples in the specified medium. The bottom and top of the colored box correspond to the observed 25th percentile and 75th percentile of motility percent values, respectively. This interval is also called the inter quartile range (IQR). The vertical lines extending from the box indicate the range of observed values within a distance of 1.5 times the length of the IQR from the median. Motility percent appears to be similarly distributed across the three media at baseline, but as time progresses, the motility percent declines more in QUINN and SPWASH than it does in MHM.

The estimated differences in mean motility percent at 8 hours and at 48 hours, comparing samples of sperm preserved in QUINN to those in MHM and comparing samples preserved in SPWASH to those in MHM are shown in Table 2. Each estimate is accompanied by a 95% confidence interval for the true difference between mediums as well as a p value quantifying the strength of evidence in the data supporting the difference. For example, at 8 hours, the mean motility percent in QUINN is estimated to be 7.12 lower than the mean motility percent in MHM, comparing samples with the same baseline motility percent. We are 95% confident that the true difference in motility percent between the mediums (QUINN minus MHM) at 8 hours is within the interval -10.57 to -3.68. The p value for the statistical test that the true difference is nonzero is p = 1:62 x 10-4: This value estimates the probability that a difference between mediums of -7.12 would be observed if indeed there were no difference in motility percent between the mediums, and the difference in observed values were only due to random chance. A very small p value indicates that the data contain strong evidence that the mean motility percent after 8 hours in QUINN is different from, and in this case lower than, that in MHM. Typically, a p value of less than 0.05 is interpreted as a statistically significant association. The values in the table for the difference in motility percent between QUINN and MHM at 48 hours and for the difference in mean motility percent between SPWASH and MHM at 8 hours and at 48 hours may be interpreted analogously.

For example, at 48 hours, the mean motility percent in SPWASH is estimated to be 17.14 lower than the mean motility percent in MHM, comparing samples with the same baseline motility percent, with a 95% confidence interval of (-21.71, -12.57) for the true difference and a p value p = 2:58 x 10-9: Box plots comparing the distributions of rapid progression percent in MHM, QUINN, and SPWASH at baseline and at 8, 24, and 48 hours are shown in Figure 2. As was observed for overall motility percent, the rapid progression percent appears to be similarly distributed for each of the three media at baseline. At each subsequent follow-up time, the rapid progression percent appears to decline more in QUINN and SPWASH than it does in MHM.

The estimated differences in mean rapid progression percent at 8 hours and at 48 hours, comparing samples of sperm preserved in QUINN to those in MHM and comparing samples preserved in SPWASH to those in MHM are shown in Table 3. Each estimate is accompanied by a 95% confidence interval for the true difference between mediums and a p value quantifying the strength of evidence in the data supporting the difference. The interpretations of the results of the linear regression in Table 3 are analogous to those shown in Table 2. For example, at 48 hours, the mean rapid progression percent in QUINN is estimated to be 17.04 lower than that in MHM, comparing samples with the same baseline rapid progression percent. The 95% confidence interval for the true difference in rapid progression percent between QUINN and MHM is (-21.65, -12.44). The p value associated with this estimate is p = 3:38 x 10-9, which indicates very strong evidence in the data supporting the difference in mean rapid progression in QUINN and MHM. As for motility percent above, differences of substantial magnitude and strong statistical significance were observed for rapid progression percent at 8 hours and at 48 hours, between QUINN and MHM and between SPWASH and MHM.

Discussion

Replicating physiological homeostasis minimizes cellular stress during routine sperm handling, generating optimal outcomes for assisted reproductive procedures, such as: 1U1, 1CS1, and 1VF [2]. During insemination, gametes and embryos are readily maintained at ideal temperature, humidity, and CO2 levels. The CO2 gas concentration regulates external pH. pHe is easily manipulated and measured; therefore, optimal pHe has been extensively examined for embryo and gamete culture. However, handling, processing, and preparation steps are typically performed outside of an incubator, where sperm are exposed to atmospheric conditions, and the risk of even minor changes in pHe can lead to undesirable outcomes in sperm viability and performance, and by extension fertility outcomes [3].

Zwitter ionic buffers, such as HEPES and MOPS, can act as either an acid or a base and are commonly used to stabilize pHe in cell culture media. It has been shown that pHe influences cells in culture through its impact on intracellular pH (pHi; 4,5). pHi in turn, regulates a variety of cellular processes including enzymatic activity, cell division, differentiation, membrane transport, protein synthesis, cell communication, cytoskeleton elements and microtubule dynamics [3]. Cells contain intrinsic pHi regulatory mechanisms, (HCO3-/Cl- exchanger, Na+/H+ antiporter, and Na+ dependent HCO3"/Cl- exchanger); however, in culture, pHi initially follows the external pH of media. For clinical use, buffers should be stable, easily prepared, and consistent lot-to-lot. HEPES and MOPS can be obtained as a free acid, or conjugated to various salts; like sodium and potassium. Combining free acid and salt forms of the same buffer in various ratios offers the ability to adjust the working pHe of the medium during formulation, without the need to adjust with an acid or base later, improving consistency of the media formulation [4].

Multipurpose Handling Media (MHM, Irvine Scientific) combines HEPES and MOPS for an improved formulation that does not require the use of a CO2 incubator to maintain physiological pH 7.2-7.4 and osmolality over a broad temperature range. Additionally, MHM solution also contains glycine and taurine to maintain cellular homeostasis through regulation of pHi [5]. Combining HEPES and MOPS for embryo culture demonstrated that the use of both buffers provide buffering at a point between HEPES or MOPS alone, and yielded similar blastocyst formation and cell number compared to the individual buffers [6]. Furthermore, preliminary studies suggest that a combination buffer of HEPES/MOPS may be useful for procedures such as ICSI, as it yielded similar rates of normal fertilization of human oocytes (71%) abnormal fertilization (5%) and blastocyst development (74%) as media buffered with HEPES alone (63%, 12%, 52%, respectively; 6).

Human sperm bioassays are highly sensitive when both motility and the quality of sperm motility (motility grade) are taken into consideration [7]. The American Association of Bioanalysts (AAB) chose an assay time of 48 hours for identifying the quality of ART culture media, we used 48 hours as an endpoint to be consistent with this guidance, and assayed motility percent and rapid progression percent, to sensitively quantify causal association of sperm performance to handling medium. The experimental design of this study enabled the causal association between handling medium and rapid and total sperm motility percent. However, it is important to carefully assess whether any unmeasured factors or factors not analyzed could explain part or all of the observed differences in motility percent and rapid progression percent between the handling media. Precise handling of cells and supplies within the lab, as well as tedious attention to detail are just as important as the media formulation to outcomes. We took care to minimize any differences across media in the handling of samples throughout the experiment and in the measurement tools or procedures used at baseline or during follow-up, so as not to introduce bias into the data.

Conclusion

Differences of substantial magnitude and strong statistical significance were observed for mean motility percent between QUINN and MHM, and between SPWASH and MHM, at both the analyzed follow-up times. Here for the first time, we have demonstrated superior performance of a commercially available dual buffer solution of HEPES and MOPS for human sperm handling. Sperm viability, motility percent, and rapid progression in MHM displayed significantly better performance than single buffer controls, presumably due to synergistic pHe and pHi stabilization afforded by a dual-buffered system.

[uberstrainer]

Streamline Your Cell Separation with pluriMate: The Ultimate Tool for Efficient Antibody Cell Separation

This blog explores the pluriMate's capabilities for antibody cell separation, highlighting its efficient cell separation, ease of use, and versatility in laboratory settings.

Antibody cell separation is a critical technique in various scientific and medical disciplines, enabling researchers to isolate specific cell populations for further analysis. While traditional methods like density gradient centrifugation have been employed for years, they often involve complex procedures and increase the risk of contamination. The need for efficient and reliable cell separation solutions has led to the development of innovative technologies, such as the pluriMate system.

How pluriMate Enhances Antibody Cell Separation

Antibody cell separation involves isolating specific cell types from a mixture, which is critical in various research and clinical applications. The pluriMate redefines this process by leveraging its unique design to streamline the separation of leukocytes and PBMCs. Traditional methods often require meticulous layering of sample material over a density gradient medium, a task that can be both time-consuming and prone to errors. However, with pluriMate, this cumbersome step is eliminated.

The device's porous barrier, made from high-grade polyurethane, prevents the direct mixing of sample material with the separation medium. This allows for a straightforward pouring of anticoagulated blood or bone marrow directly into the pluriMate tube. During centrifugation, the device efficiently separates leucocytes, lymphocytes, and PBMCs based on their density, utilizing various density gradients like Leuko Spin, Lympho Spin, and PLT Spin. The result is a clean separation with the enriched cell fraction residing in an interphase above the separation medium, safeguarded from contamination during harvest.

Key Features and Benefits

Simple Filling Process

One of the standout features of pluriMate® is its simple filling process. Unlike conventional methods that require careful layering of blood over the separation medium, pluriMate eliminates this step. This design innovation not only reduces the risk of contamination but also streamlines the preparation process, saving valuable time in the laboratory.

Reduced Contamination Risk

With pluriMate, the risk of contamination is significantly lowered. The integrated porous sponge acts as a barrier that prevents sample material from mixing with the separation medium. This ensures that the final cell fraction remains pure and free from unwanted cell types or debris, which is crucial for accurate and reliable results in antibody cell separation.

Versatility with Cell Separation Techniques

The pluriMate is compatible with various cell separation techniques and can be used in conjunction with products like pluriSpin. By simply adding the pluriSpin solution to the sample, incubating, and spinning down, researchers can achieve efficient negative cell separation. This versatility makes pluriMate a valuable asset for a wide range of applications, from basic research to advanced clinical studies.

Pre-filled and Unfilled Options

pluriMate offers both pre-filled and unfilled options. The pre-filled version minimizes preparation effort and ensures consistency in the density gradient medium. For those who prefer to customize their gradients or work with specific protocols, the unfilled option provides flexibility while still benefiting from the device's advanced separation capabilities.

Applications of pluriMate in Research

The pluriMate is designed to meet the demands of diverse research and industrial applications. In research settings, it facilitates the rapid and accurate separation of target cells, essential for studies involving cell function, genetic analysis, and more. In clinical laboratories, it supports efficient processing of blood samples for diagnostic purposes.

How to Use pluriMate for Optimal Results

To achieve optimal results with pluriMate®, follow these steps:

Prepare the Sample: Anticoagulated blood or bone marrow should be collected and ready for processing.

Fill the Tube: Pour the sample directly into the pluriMate tube, taking advantage of its simple filling process.

Centrifuge: Perform centrifugation based on the selected density gradient protocol (e.g., Leuko Spin).

Harvest Cells: After centrifugation, carefully collect the enriched cell fraction from the interphase, protected from contamination by the porous sponge.

Conclusion

The pluriMate is a powerful tool in the realm of antibody cell separation and cell enrichment techniques. Its innovative design simplifies the cell separation process, reduces contamination risk, and offers versatility across various applications. By integrating pluriMate into your laboratory workflows, you can achieve more efficient and reliable results, making it an indispensable asset for researchers and businesses dedicated to advancing cellular studies.

[realdrake]

Purifying nanoparticles

Purifying nanoparticles into monodisperse fractions is a crucial step in understanding their physical properties and minimizing variability in biological applications. The lack of uniformity in nanoparticle samples can lead to inconsistent results, making it challenging to draw meaningful conclusions. While various methods have been explored for purifying "hard" nanoparticles, few studies have focused on "soft" nanoparticles like polymersomes.

Polymersomes, with their unique physico-chemical and mechanical properties, are attractive for biological and medical applications. However, their self-assembly process inherently leads to broad size distributions and high variability in morphology. This heterogeneity can result in a mixture of spherical vesicles, tubes, and genus structures, as well as non-vesicular structures like micelles. To address this challenge, we compared four complementary techniques for separating polymersomes by size and shape: cross-flow filtration (CFF), differential centrifugation (DC), size exclusion chromatography (SEC), and density gradient centrifugation (DGC).

the  results show that each technique has its advantages and disadvantages. CFF efficiently separates micelles from polymersomes, possibly due to a combination of size exclusion and differential fluid dynamics. However, it cannot be used to separate sub-populations of polymersomes by size. DC and SEC enable separation of polymersomes into distinct size fractions, but result in sample concentration loss. DGC, on the other hand, achieves shape-based separation by exploiting differences in membrane packing density.

they found that the different shape of polymersomes corresponds to changes in the density of membrane packing, providing the means for their separation by the DGC-based method. This approach allows for the separation of polymersomes into distinct fractions based on their shape, which is essential for understanding their physical properties and biological behavior.

By combining these techniques, we can develop efficient purification protocols for polymersomes and other nanoparticles. This study demonstrates the importance of purification in producing consistent and reproducible results in biological experiments and improving application development in medicine and drug delivery. The ability to purify nanoparticles into monodisperse fractions will enable researchers to better understand their physical properties and behavior, ultimately leading to the development of more effective nanoparticle-based therapies.

[pragya1-blog]

Bone Marrow Aspirate Concentrates Market is Expanding at a CAGR of 5.0% by 2025

Concentrated bone marrow aspirate (BMA) is a biologic concentrate derived from a patient’s own bone marrow, which represents 0.001-0.01% mononuclear cell concentration, red blood cell, immature myeloid precursors, granulocytes, and platelets. Gradient centrifugation is applied to BMA to separate mononuclear cells, (also known as BMAC) which can be used for the treatment of bone healing, cartilage repair and new blood vessel growth. The global bone marrow aspirate concentrates market was valued around US$ 130 Million and expected to reach over US$ 200 Million registering a growth rate more than 5.0% during forecast period of 2017 to 2025. Rising demand for technology driven solution, rapid economic growth, increasing disposable income, and growing awareness about stem cell-based treatment in the region have generated more opportunity, creating potential market from players in the U.S. and Europe.

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https://www.transparencymarketresearch.com/bone-marrow-aspirate-concentrates-market.html

Technological advancements in the bone marrow aspirate concentrates systems segment will drive growth of the segment in near future. Technological advancement is the development of a better device (in terms of BMAC preparation time, quantity of bone marrow stem cells separated, and cost) than the existing one. For instance, the ‘Harvest SmartPrep BMAC system’ is a point-of-care, easy-to-use system capable of separating a defined concentration of mononucleotide cells within 15 minutes. On the other hand, the existing centrifugation method is labor-intensive and time-consuming. It requires complex logistic considerations. Thus, technologically advanced BMAC solutions are anticipated to replace current procedure in near future. Emergence of biologic alternatives like PRP, BMAC and stem cell derived products are about to set a trend in the current market scenario as the orthobiologic procedures are considered to be less invasive and more effective as compared to traditional implantation procedures.

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With the advancement in production protocol and equipment, BMAC has demonstrated positive results in several cases. After the discovery of bone marrow therapy, the treatment has been associated with painful outcomes and prolonged recovery time. Sometimes, the treatment does not show any positive therapeutic improvement in pain management and wound healing time. Thus, high failure rate has been observed during clinical research trials for dermatological application which may hamper growth of the market. The bone marrow aspirate concentrates market in Asia is expanding with a high potential and has created huge opportunities owing to unmet clinical needs, rising geriatric population, large patient pool, favorable government regulations, development in health care sector, and increased focus on research and developmental activities. Companies operating in the global market for bone marrow aspirate concentrates are focusing on in-licensing and collaboration agreements to put new products in the Asia market.

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[newdayessays]

Explain the difference between negative and positive selection isolation methods according to MACS protocols.

Explain the difference between negative and positive selection isolation methods according to MACS protocols.

1st Assignment (30 Points)

Covers Material Presented in Units 1-3

Section I. (20 Points Total)

The following Topics 1-3 (of 4) cover the cell isolation protocols from Units 1 & 2: Assigned Appendix readings; Isolation of human monocytes by double gradient centrifugation; the Rosette Sep ™ isolation method; and protocols using MACs technology. Your answers should explain the main techniques in…

[uberstrainerusa]

TwinSpin and centrifugation: How it works?

TwinSpin centrifuge tubes support density gradient centrifugation. Let’s find out how and we will also discuss the protocol for the use of TwinSpin in combination with density gradient media.

Scientists divide materials using the centrifugation technique based on their size and shape. The two types of centrifugal methods used to separate particles are differential centrifugation and density gradient centrifugation. This blog will discuss TwinSpin's functionality as well as how it supports density gradient centrifugation.

Peripheral blood mononuclear cells (PBMC) can be separated from whole blood and bone marrow using TwinSpin centrifuge tubes that have already been pre-filled with PBMC Spin Medium. The TwinSpin is made up of an inner tube and a standard 15. The inner tube's open bottom is buried in the density gradient medium (DGM). The blood sampling tube can be directly pipetted into the TwinSpin with anticoagulated blood or bone marrow. 

The sample is placed on top of the DGM inside the inner tube. Depending on the density gradient used (Leuko Spin, PBMC Spin, PBMC 24+, or PLT Spin Medium), leukocytes, lymphocytes, and PBMCs are separated from unwanted erythrocytes and granulocytes during Density Gradient Centrifugation. 

Target cells above the DGM are therefore enriched in the interphase. Through the DGM, the erythrocytes will exit the inner tube and fall to the bottom of the outer tube. Once the inner tube has completely separated, just take it out. 

As a valve, the elastic cap is used. The collection tube can be converted into a pipette by removing the cap. Drop by drop, the contents can be collected.

Instructions for Using the TwinSpin Tube

1. Ensure that the (diluted blood) sample, the TwinSpin, and the centrifuge are all at room temperature.

1.1 Check that the inner tube is partially filled and immersed in the DGM. If not, maintain the vertical position while rotating the TwinSpin device.

1.2 Take out and throw away the transport stopper

2. Include Sample Material

2.1 Pipette the sample material on top of the DGM in the inner tube by tilting the TwinSpin 45° and allowing the sample to run down the inside of the dropper.

2.2 Push the elastic cap firmly into the opening to close the TwinSpin. PluriSpin PLT Depletion can be used to reduce platelet contamination.

3. Spin

3.1 Centrifuge at 800 x g for 20 minutes at room temperature in a swing bucket rotor, brake off. If the blood is more than 4 hours old, centrifuge it for 30 minutes at 1000g.

4. Collect

4.1 Remove the inner separation tube by unscrewing it.

4.2 Push the elastic cup down to collect cells in the opaque layer in a new tube.

5 Wash (if necessary)

5.1 Add wash buffer to the reaction tube.

5.2 Spin down cells for 10 minutes at 4°C with 300 x g (no or small brake).

5.3 Remove the supernatant and set the reaction tube on the table for 20 seconds.

Excess wash buffer will flow down the tube wall and accumulate at the bottom. Aspirate the majority of the liquid above the pellet. The liquid will appear foggy because it contains mostly platelets; aspiration will improve the washing result.

5.5 Carefully pipette the pellet with 1 ml of wash buffer.

5.6 Re-do steps 5.1–5.4.

5.7 Refill the pellet to the desired volume.

Want to see TwinSpin in action? Check out our line of products that supports density gradient centrifugation, and let us know if you have any questions.

[abbkineeu]

New Post has been published on Biotech Advisers

New Post has been published on http://www.bioadvisers.com/immunoprecipitation/

Immunoprecipitation

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or extrinsically labeled cells, subcellular fractions from either unlabeled or labeled cells, or in vitro–translated proteins. Immunoprecipitation is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or sedimentation on density gradients. Either polyclonal or monoclonal antibodies from various animal species can be used in immunoprecipitation protocols. Antibodies can be bound noncovalently to immunoadsorbents such as protein A– or protein G–agarose, or can be coupled covalently to a solid-phase matrix.

Figure 1 Schematic representation of the stages of a typical immunoprecipitation protocol

Immunoprecipitation protocols consist of several stages (Fig 1). In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture or as a monolayer on tissue culture dishes with nondenaturing detergents. Cells can also be lysed under denaturing conditions. Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents. All of these lysis procedures are suitable for extracting antigens from animal cells. Two commonly used buffers for cell lysis: RIPA buffer gives lower background in IP. However, RIPA can denature some proteins. If you are conducting IP experiments to study protein-protein interactions, RIPA should not be used as it can disrupt the interactions; NP-40 buffer denatures proteins to a lesser extent, and is thus used for phosphorylation experiments when studying kinase activity. NP-40 is typically used for the study of protein-protein interactions. NP-40 is a nonionic detergent and the most commonly used detergent in cell lysis buffers for IP and westerns. Yeast cells require disruption of their cell wall in order to allow extraction of the antigens.

In stage 2, a specific antibody is attached, either noncovalently or covalently, to a sedimentable, solid-phase matrix to allow separation by low-speed centrifugation. For example, the noncovalent attachment of antibody to protein A or protein G agarose beads. Incubation will depend upon the concentration of target protein and the specificity of the Ab toward this target (Table 1).

Table 1 Binding characteristics of different immunoglobulins (Igs).

++, moderate to strong binding; +, weak binding; −, no binding

Stage 3 consists of incubating the solubilized antigen from stage 1 with the immobilized antibody from stage 2, followed by extensive washing to remove unbound proteins. Immunoprecipitated antigens can be dissociated from antibodies and reprecipitated by a protocol referred to as “immunoprecipitation-recapture”. This protocol can be used with the same antibody for further purification of the antigen, or with a second antibody to identify components of multisubunit complexes or to study protein-protein interactions. Immunoprecipitated antigens can be analyzed by one-dimensional electrophoresis, two-dimensional electrophoresis, or immunoblotting. In some cases, immunoprecipitates can be used for structural or functional analyses of the isolated antigens. Immunoprecipitates can also be used as sources of immunogens for production of monoclonal or polyclonal antibodies.

[bioadvisers]

Bioadvisers shared on Biotech Advisers

Immunoprecipitation

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or extrinsically labeled cells, subcellular fractions from either unlabeled or labeled cells, or in vitro–translated proteins. Immunoprecipitation is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or sedimentation on density gradients. Either polyclonal or monoclonal antibodies from various animal species can be used in immunoprecipitation protocols. Antibodies can be bound noncovalently to immunoadsorbents such as protein A– or protein G–agarose, or can be coupled covalently to a solid-phase matrix.

Figure 1 Schematic representation of the stages of a typical immunoprecipitation protocol

Immunoprecipitation protocols consist of several stages (Fig 1). In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture or as a monolayer on tissue culture dishes with nondenaturing detergents. Cells can also be lysed under denaturing conditions. Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents. All of these lysis procedures are suitable for extracting antigens from animal cells. Two commonly used buffers for cell lysis: RIPA buffer gives lower background in IP. However, RIPA can denature some proteins. If you are conducting IP experiments to study protein-protein interactions, RIPA should not be used as it can disrupt the interactions; NP-40 buffer denatures proteins to a lesser extent, and is thus used for phosphorylation experiments when studying kinase activity. NP-40 is typically used for the study of protein-protein interactions. NP-40 is a nonionic detergent and the most commonly used detergent in cell lysis buffers for IP and westerns. Yeast cells require disruption of their cell wall in order to allow extraction of the antigens.

In stage 2, a specific antibody is attached, either noncovalently or covalently, to a sedimentable, solid-phase matrix to allow separation by low-speed centrifugation. For example, the noncovalent attachment of antibody to protein A or protein G agarose beads. Incubation will depend upon the concentration of target protein and the specificity of the Ab toward this target (Table 1).

Table 1 Binding characteristics of different immunoglobulins (Igs).

++, moderate to strong binding; +, weak binding; −, no binding

Stage 3 consists of incubating the solubilized antigen from stage 1 with the immobilized antibody from stage 2, followed by extensive washing to remove unbound proteins. Immunoprecipitated antigens can be dissociated from antibodies and reprecipitated by a protocol referred to as “immunoprecipitation-recapture”. This protocol can be used with the same antibody for further purification of the antigen, or with a second antibody to identify components of multisubunit complexes or to study protein-protein interactions. Immunoprecipitated antigens can be analyzed by one-dimensional electrophoresis, two-dimensional electrophoresis, or immunoblotting. In some cases, immunoprecipitates can be used for structural or functional analyses of the isolated antigens. Immunoprecipitates can also be used as sources of immunogens for production of monoclonal or polyclonal antibodies.

[abbkine]

BioAdvisers said on Biotech Advisers

Immunoprecipitation

Immunoprecipitation is a technique in which an antigen is isolated by binding to a specific antibody attached to a sedimentable matrix. The source of antigen for immunoprecipitation can be unlabeled cells or tissues, metabolically or extrinsically labeled cells, subcellular fractions from either unlabeled or labeled cells, or in vitro–translated proteins. Immunoprecipitation is also used to analyze protein fractions separated by other biochemical techniques such as gel filtration or sedimentation on density gradients. Either polyclonal or monoclonal antibodies from various animal species can be used in immunoprecipitation protocols. Antibodies can be bound noncovalently to immunoadsorbents such as protein A– or protein G–agarose, or can be coupled covalently to a solid-phase matrix.

Figure 1 Schematic representation of the stages of a typical immunoprecipitation protocol

Immunoprecipitation protocols consist of several stages (Fig 1). In stage 1, the antigen is solubilized by one of several techniques for lysing cells. Soluble and membrane-associated antigens can be released from cells grown either in suspension culture or as a monolayer on tissue culture dishes with nondenaturing detergents. Cells can also be lysed under denaturing conditions. Soluble antigens can also be extracted by mechanical disruption of cells in the absence of detergents. All of these lysis procedures are suitable for extracting antigens from animal cells. Two commonly used buffers for cell lysis: RIPA buffer gives lower background in IP. However, RIPA can denature some proteins. If you are conducting IP experiments to study protein-protein interactions, RIPA should not be used as it can disrupt the interactions; NP-40 buffer denatures proteins to a lesser extent, and is thus used for phosphorylation experiments when studying kinase activity. NP-40 is typically used for the study of protein-protein interactions. NP-40 is a nonionic detergent and the most commonly used detergent in cell lysis buffers for IP and westerns. Yeast cells require disruption of their cell wall in order to allow extraction of the antigens.

In stage 2, a specific antibody is attached, either noncovalently or covalently, to a sedimentable, solid-phase matrix to allow separation by low-speed centrifugation. For example, the noncovalent attachment of antibody to protein A or protein G agarose beads. Incubation will depend upon the concentration of target protein and the specificity of the Ab toward this target (Table 1).

Table 1 Binding characteristics of different immunoglobulins (Igs).

++, moderate to strong binding; +, weak binding; −, no binding

Stage 3 consists of incubating the solubilized antigen from stage 1 with the immobilized antibody from stage 2, followed by extensive washing to remove unbound proteins. Immunoprecipitated antigens can be dissociated from antibodies and reprecipitated by a protocol referred to as “immunoprecipitation-recapture”. This protocol can be used with the same antibody for further purification of the antigen, or with a second antibody to identify components of multisubunit complexes or to study protein-protein interactions. Immunoprecipitated antigens can be analyzed by one-dimensional electrophoresis, two-dimensional electrophoresis, or immunoblotting. In some cases, immunoprecipitates can be used for structural or functional analyses of the isolated antigens. Immunoprecipitates can also be used as sources of immunogens for production of monoclonal or polyclonal antibodies.