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Yes, it is possible to perform CUT&RUN on primary cells. We have shown that our CUT&RUN Assay Kit #86652 works with 100,000 human CD8+ live T cells with antibodies against epigenetic regulators and transcription factors.

Histone Methylation

Expert-reviewed interactive pathway providing a current overview of Histone Methylation.

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If you start with 100,000 cells, you typically get 0.6-6ng DNA. However, if you start with 5,000 - 10,000 cells, the DNA yield can be as low as 0.1-0.2 ng or even undetectable by the picogreen assay. In this case, we suggest you directly proceed with library prep using 20 PCR amplification cycles, which will result in a library DNA with a concentration of around 10-30 ng/ul. QC of the CUT&RUN DNA by qPCR can be performed on the library DNA if you hope to save as much CUT&RUN DNA as possible for the library prep.

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Specific activity (expressed in units/mg) is a function of cytokine or growth factor purity and appropriate protein folding. To determine the specific activity, typically the readout of cytokine treatment is measured in a standardized bioassay. The ED50 (expressed in ng/ml), which is the concentration of cytokine or growth factor required to induce half the maximal biological response, is determined in the assay. The following formula can be used to convert the activity as an ED50 in ng/ml to specific activity in units/mg:
Specific activity (units/mg) = 10E6 / ED50 (ng/ml).
For example, the ED50 for our Human Leukemia Inhibitory Factor (hLIF) #8911 is approximately 0.85ng/ml (based on the median ED50 range from the webpage/datasheet) and therefore the specific activity would be 1.17 X 10^6 Units/mg:
10ug = 11,700 Units
50ug = 58,500 Units

The Potential of Proteomics: A Clear Path to Discovery

Whether your lab is equipped to do Mass Spec analysis in-house or has never considered setting up a proteomics experiment before, the Proteomics group can help.

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Our tissue extracts are prepared as follows:

Cut a small piece of tissue (approximately 3 mm^3).

Place in a clean Dounce homogenizer.

Add 1mL cold 1X Cell Lysis Buffer #9803 + 1.0 mM PMSF #8553 + 1X Protease/Phosphatase Inhibitor Cocktail #5872.
Homogenize well (approximately 20 strokes).
Sonicate 3 times for 15 seconds each (cool on ice in between).
Microcentrifuge at 12,000 rpm for 15-20 minutes to remove insoluble debris.
Add 333uL of 3X SDS Sampler Buffer (adjust as needed depending on how much 1X Cell Lysis Buffer you added) or store at -80C in 1X Cell Lysis Buffer.

Notes:

Keep unused tissue on dry ice.

Romantic Reagents

Use antibody vials instead of candy hearts!?! That's crazy...or is it?

Lifetime Achievement Award

Cell Signaling Technology CEO and Founder Dr. Michael Comb wins lifetime achievement award.

Hallmarks of Validation - Ranged Strategy

A ranged validation strategy includes both endogenous and heterologous models that express high, moderate, and low levels of the target of interest.

Angiogenesis Resources

Overview of Angiogenesis antibodies & related reagents, interactive pathway diagrams, and other technical resources.

Mitochondrial Control of Apoptosis

Explore the mitochondrial apoptosis pathway and its role in programmed cell death. Read more about this essential pathway here.

AMPK Signaling

Explore the AMPK pathway and its role in cellular energy homeostasis. Uncover the mechanisms of energy regulation. Learn more here.

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Our β-Actin (13E5) Rabbit mAb #4970 detects endogenous levels of total β-actin protein. Despite the high sequence identity between the cytoplasmic actin isoforms, β-actin and cytoplasmic γ-actin, our β-Actin (13E5) Rabbit mAb #4970 does not cross-react with cytoplasmic γ-actin, or any other actin isoforms.

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PI4 Kinase Antibody #4902 detects endogenous levels of total PI4 Kinase isoform 230. In humans, PI4K230, PI4K92, and PI4K55 are four PI4 kinase isoforms that have been characterized and classified according to their molecular weights of 230, 92, and 55 kDa. In the literature, alpha refers to the 230 kDa size band (UniProt ID: P42356) and beta refers to the 92 kDa size band (UniProt ID:Q9UBF8). Type 2 alpha (UniProt ID: Q9BTU6 ) and type 2 beta (UniProt ID: Q8TCG2) refer to the 55 kDa band.

MAP Kinase Signaling Resources

Overview of MAP Kinase signaling pathways, antibodies and related reagents, interactive pathway diagrams, and other technical resources.

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LC3 exists as three highly homologous isoforms or paralogs. These are LC3A, LC3B, and LC3C. These LC3 paralogs have different patterns of expression in human tissues [see He, H et al. (2003) J Biol Chem 278(31), 29278-87 (PMID 12740394 Figure 2A; http://www.ncbi.nlm.nih.gov/pubmed/12740394)] and undergo post-translational modifications during autophagy. Cleavage of LC3 (i.e., LC3A, LC3B, and/or LC3C) at the carboxy terminus immediately following synthesis yields the cytosolic LC3-I form. During autophagy, LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 that allows for LC3 to become associated with autophagic vesicles. The presence of LC3 in autophagosomes and the conversion of LC3 to the lower migrating form LC3-II are used as markers of autophagy.

Cellular Metabolism

Overview of Cellular Metabolism pathways, antibodies and related reagents, interactive pathway diagrams, and other technical resources

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While we do not specifically test our antibodies using co-IP, we expect IP-approved antibodies to work well for co-IP experiments. As each protein-protein interaction is unique, optimization of lysis conditions and the inclusion of proper controls are necessary for a successful experiment. More information on experimental design for IP and co-IP experiments can be found here.

For co-IPs, we suggest blotting for the target protein first to confirm pull-down. Once confirmed, blotting for additional proteins in the complex can be performed. If your protein of interest runs at a size similar to the heavy or light chain of IgG, we recommend the use of a conformation-specific or

Adhesion/ECM/Cytoskeleton

Overview of Adhesion and Extracellular Matrix signaling networks, antibodies and related reagents, interactive pathway diagrams, and technical resources.

Tissue Clearing from a Microscope Enthusiast’s Perspective

Biological imaging data has been increasing in complexity. This opens up the opportunity for a host of insights regarding spatial relationships.

The Reproducibility Crisis: An open letter from CST's Chief Scientific Officer

The time is now for the scientific community to come together to respond to the challenges of reproducibility and to be part of the solution.

20 Years of Research, Reagents, and Lasting Relationships

Today marks 20 years of impact in scientific research and community support. Thank you for standing with us.

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We use Microcon columns from Amicon (30 kDa cut off) to concentrate media. It is important that the medium does not contain FBS because this will make it difficult to concentrate with the Microcon columns.
1. Spin the media (20 ml) to pellet any cellular debris.
2. Add the 20ml volume to a Microcon column and concentrate it down to 500 ul.
3. Add 10 ml of cold 1xPBS to the 500 ul sample to wash it and concentrate again to 500 ul.
4. Transfer the 500 ul of concentrated media to an eppendorf tube and wash the Microcon tube membrane twice with 250 ul of cold 1xPBS to remove any proteins stuck to the membrane. This should result in a 1ml sample.
5. Add 500 ul of 3x reducing SDS sample buffer to 1.0 ml of concentrated media. MMPs tend to be quite unstable and degrade easily in our hands, so we highly suggest adding protease and phosphatase inhibitors to the lysate, along with PMSF and 5 mM EDTA (as a chelator).
6. Aliquot the lysates and store them at -80C.

Inhibition of Apoptosis

Expert-reviewed interactive pathway providing a current overview of Inhibition of Apoptosis Signaling.

Characterize Myeloid Cell Mediated Immunosuppression in the Tumor Microenvironment Using Multiplex IHC

Explore the protocol and technical considerations for selecting and using antibodies in mIHC

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There may be some lot-to-lot variability in the signal strength of our Vimentin (V9) Mouse mAb #49636 when testing by western blot due to this antibody being sold by weight instead of reactivity.

Erk Signaling

Phospho-Erk product lists, product citation lists, product selection tools, comparison tables and educational resources for MAPK signaling from Cell Signaling Technology.

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We recommend a 1 hour incubation at room temperature. We have found that prolonged incubation (>2hrs) and elevated incubation temperatures (>25C) can promote non-specific binding.

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The top band in the gel image for our Human ACE2 (18-652) Recombinant Protein (mFc-Tag) #83986 likely represents dimerization due to the mFc tag.

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This decision was based on our analysis of hundreds of CUT&RUN samples. We found that a sequencing depth lower than 3 million generated too few peaks and that the number of peaks increased with deeper sequencing. If desired, one can increase the sequencing depth to 10 million reads per sample. The duplication rate of sequencing reads significantly increases if the sequencing depth is >15 million reads per sample.