Technical Support Articles

Why doesn't PD-L2 migrate at 31 kDa?

The predicted molecular weight of PD-L2 is 31 kDa (based on amino acid composition). However, in most models, PD-L2 migrates at an observed molecular weight of 45-60 kDa on Tris-Gl...

My Tris-Glycine SDS Running Buffer (10X) #4050 has turned yellow. Is it still okay to use?

It is normal for the Tris-Glycine SDS Running Buffer (10X) #4050 to turn yellow as it ages. The yellowing has no effect on the product's performance and is no cause for concern...

What DNA library prep kit do you recommend using for the CUT&Tag assay?

What can I use as a loading control with the Cell Fractionation Kit #9038?

Due to the nature of the Cell Fractionation Kit #9038 and the localization of each of the proteins in the Loading Control Sampler Kit #5142, the proteins will not be equally expres...

Does the CST CUT&Tag kit work with isolated nuclei?

Our CUT&Tag kit is optimized for use with whole cells and works with a large number of cell lines, both adherent and suspension. While our kit works with pre-isolated nuclei be...

What is the best way to choose an antibody for my CUT&Tag assay if I can not find a CUT&Tag-validated antibody for my target protein?

We always recommend starting with CST CUT&Tag-validated antibodies because CST scientists perform intensive in-house tests to exclude any antibody that tends to show non-specif...

How many assays can be performed with the Senescence β-Galactosidase Staining Kit #9860?

The reagents provided with our Senescence β-Galactosidase Staining Kit #9860 are sufficient to stain 125 x 35 mm wells. The volumes listed in our protocol are intended for one ...

Which species does the Caspase-3 Activity Assay Kit #5723 work in?

We have validated the Caspase-3 Activity Assay Kit #5723 in human and mouse cell lines. However, as long as there is active caspase-3 present, this kit is predicted to work with mo...

Will the RAG1 (D36B3) Rabbit mAb #3968 cross-react with human RAG2?

Our RAG1 (D36B3) Rabbit mAb #3968 was produced using a recombinant protein fragment with a sequence toward the N-terminal region of human RAG1. No significant homology is found in ...

Can I freeze the PTMScan® HS Magnetic Immunoaffinity Beads?

The PTMScan HS Magnetic Immunoaffinity Beads should not be frozen. We recommend storing this product at 4C. However, we have performed functional testing by IAP and subsequent mass...

How do I pool CUT&Tag DNA libraries with a variety of yields?

Usually, the CUT&Tag DNA libraries from histone targets have a higher concentration than those from non-histone targets. We use the following formula to convert a library conce...

Why is the PTMScan® LysC Protease #84748 digestion performed at room temperature and not at 37°C?

The digestion is performed at room temperature to minimize carbamylation (also known as carbamoylation) of the sample peptides in the urea buffer, which can interfere with downstre...

Why do I see a doublet when using my total Histone H3 antibody for western blot?

We see this doublet phenomenon often in a number of different cell lines that we test with our Histone H3 antibodies. It does appear to be sample dependent. The doublet may be due ...

Why is there an additional band at 24kDa using the Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718?

The extra band(s) around 24 kDa using our Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb #9718 are most likely mono-ubiquitylated H2AX. H2AX, like H2A, is mono-ubiquitylated.

How long can I leave glycerol on my plates for the Senescence β-Galactosidase Staining Kit #9860?

From our limited in-house testing, we would suggest leaving glycerol on the plates for 3-5 days at most. We have not tested longer time periods than this.

How many LC-MS/MS injections are obtained from a PTMScan® enrichment?

Each PTMScan® enrichment contains sufficient peptides for three to four LC-MS/MS runs. Therefore, one enrichment can produce enough peptides for technical replicates and enough rem...

What is the pH of SignalStain® Antibody Diluent #8112?

The pH of our SignalStain^® Antibody Diluent #8112 has a range between 7.38-7.45. 

Is it possible to perform CUT&Tag on fixed frozen cells?

Yes. Cell fixation is recommended when the cells are very fragile (easily lysed during experiments), when the cellular signaling pathway is sensitive to the Concanavalin A beads...

Why does MAFA (D2Z6N) Rabbit mAb #79737 recognize a band at approximately 48 kDa when the predicted molecular weight is approximately 37 kDa?

MAFA is a phosphoprotein. The multiple phosphorylation sites on this protein cause a mobility shift, resulting in the protein migrating slower by gel electrophoresis. This has been...

Do your Phospho-Acetyl-CoA Carboxylase (Ser79) (D7D11) Rabbit mAbs detect both the alpha and beta isoforms?

Our Phospho-Acetyl-CoA Carboxylase (Ser79) (D7D11) Rabbit mAbs #11818 and #42801 detect both phospho-ACC-alpha and phospho-ACC-beta. These antibodies recognize ACC-alpha only when ...

Can you share your protocol for preparing Jurkat -/+ Anti-CD3 antibody (10 µg/mL for 2 min) cell extracts?

We prepare Jurkat -/+ Anti-CD3 antibody (10 µg/mL for 2 min) cell extracts as follows: