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UbiScan® - Ubiquitination Proteomics

Deciphering Immuno-Oncology: Targeting Cellular Mechanisms of the Tumor Immune Response

Key signaling pathways modulate immune cell activation and tumor checkpoint evasion. Improved understanding of tumor microenvironment crosstalk and mechanisms of response/reistance will be key for next-generation cancer immunotherapies.

PTMScan® Sumoylation Remnant Motif Kit - SUMOylation Proteomics

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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 confirmed by treatment with CIAP, which has been shown to result in a shift in MAFA’s mobility from approximately 48 kDa to a more rapidly migrating form of approximately 40 kDa (see PMID: 17682063 Figure 1A; https://pubmed.ncbi.nlm.nih.gov/17682063/).

N-Terminal AcetylScan® Proteomics

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β-actin can be cleaved by caspase-3 between Asp244 and Gly245, which will result in a 32kDa fragment and a 15kDa fragment. Due to the location of its antigen, our β-Actin Antibody #4970 will detect both full-length β-actin as well as the 32kDa fragment, also known as fractin.

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Our Normal Goat Serum #5425 is not heat inactivated. If you are worried about enzymes influencing your experiment, heat inactivation is recommended. Incubating the Normal Goat Serum in a 56°C water bath for 30 minutes should be sufficient to inactivate the majority of restriction endonucleases.

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We have performed a number of comparisons over the years looking at protocol variables such as the retrieval solution, diluent, incubation, and detection methodology. We have seen that the choice of reagent in these steps can have an impact upon staining. Not every antibody is sensitive to all variables, and some antibodies will be more sensitive than others. Because we cannot perform protocol comparisons for every product that we recommend, we cannot know for certain which antibodies may be sensitive to protocol variations. For that reason, we strongly encourage customers to follow our recommended protocol when using our antibodies, including the recommended reagents, since we know that our antibodies perform well with this combination.

How to Select an Antibody for Immunoprecipitation

Protein enrichment by immunoprecipitation is frequently carried out prior to analysis. In this Tech Tip, Sarah presents considerations for choosing antibodies and beads for IP.

How CUT&RUN Profiles Chromatin

This animated explainer shows how the CUT&RUN method works for mapping protein-DNA interactions in native chromatin, while using fewer cells and saving you time.

KinomeView® Profiling Kit and Services

SignalScan™ Peptide Mix (SARS-CoV-2)

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Our COX IV (3E11) Rabbit mAb #4850 will specifically label human cells and not mouse, and our COX IV (D6I4K) Rabbit mAb (Rodent Specific) #38563 will specifically label mouse cells and not human.

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We have several antibodies for detecting the phosphorylation of ribosomal protein S6 (rpS6) at Ser235/236 that are validated for IF-IC with mouse samples. These are Phospho-S6 Ribosomal Protein (Ser235/236) Antibody #2211, Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP® Rabbit mAb #4858, Phospho-S6 Ribosomal Protein (Ser235/236) (91B2) Rabbit mAb #4857, Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mAb #4856, and Phospho-S6 Ribosomal Protein (Ser235/236) (E2R1O) Mouse mAb #62016. All of these antibodies are suitable for immunofluorescence in mouse cells.

Comparison Tables

Comparison tables for product research

The CST Hallmarks of Validation

Know what to look for and dig deeper into the validation data for your antibody to avoid the frustration of ambiguous or misleading results.

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HPSE is synthesized as a 65 kDa inactive precursor that undergoes proteolytic processing, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The 65kDa proform and the 50 kDa subunit (amino acids 158-543) are detected by the HPSE (E3N3H) Rabbit mAb #99756, while the 8kDa subunit (amino acids 36-109) is not.

Protect Your Drug Discovery Pipeline

To keep your projects moving forward, you need to count on reagents that will work as expected and be available throughout the lifetime of your study.

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Our Fc Block CD16/CD32 (2.4G2) Rat mAb #88280 binds to the Fc receptor on the surface of live cells and blocks the interaction of the Fc receptor with the Fc domain of antibodies. Preventing this interaction ensures that your antibodies will bind only to their intended target, rather than being sequestered by Fc receptors.

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We recommend using our CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 to make DNA libraries for CUT&Tag. Our DNA Library Prep Kit for Illumina (ChIP-seq, CUT&Tag) #56795, and its paired Multiplex Oligos for Illumina (Dual Index Primers) (ChIP-seq, CUT&Tag) #47538 or Multiplex Oligos for Illumina (Single Index Primers) (ChIP-seq, CUT&Tag) #29580 are not compatible with CUT&Tag DNA samples.

Illustration of Motif Antibody Coverage

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In section C, part 8 of the Senescence β-Galactosidase Staining Kit #9860 protocol, you may overlay the plates with 70% glycerol directly after removing the staining solution. It is not necessary to wash off the staining solution prior to overlaying with glycerol.

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Yes. Cell fixation is recommended when the cells are very fragile (easily lysed during experiments), when the cellular signaling pathway is sensitive to the Conconavalin A beads used for cell binding, when the target protein binds weakly to DNA, or when you hope to freeze the cell pellets for future use. A light fixation (0.1% formaldehyde for 2 min) is critical for the success of CUT&RUN with fixed cells. A reverse crosslinking step is also required at the end of the assay to remove protein from DNA. We find that live cells generally have better performance than fixed cells in the CUT&RUN assay, therefore, we recommend using live cells if possible, unless fixation is necessary as noted in the above-mentioned scenarios.

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Our total c-Myc antibodies, product numbers #9204, #5605, #18583, #89403, will recognize c-Myc1 (p67, isoform 2, 454 a.a.) and c-Myc2 (p64, isoform 1, 439 a.a.). They will not detect c-MycS (339 a.a.) or N-Myc, but there is a very small possibility that they may recognize L-Myc.

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The Caspase-4 Antibody #4450 recognizes an epitope that is present in both the full-length protein and the p20 subunit. Therefore, this antibody is predicted to detect both targets. However, we have not confirmed the reactivity of the Caspase-4 Antibody #4450 with the p20 subunit at endogenous levels. The exact protein load of recombinant, active caspase-4 shown in the western image on the product datasheet for the Caspase-4 Antibody #4450 has not been recorded. However, we typically load 1-5 ng of recombinant protein for such experiments.

CST Media Room

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We recommend that you run your samples on a 3-8% Tris-Acetate gel in order to get separation of large proteins. We also suggest that you use a standard wet transfer, transferring at 70V for 5 hours with a 0.2 um nitrocellulose membrane, in a 4C refrigerator. We also recommend reducing the methanol content in your transfer buffer to 5-10%. Using these conditions will ensure optimal and clean transfer of the RyR1 protein from the gel to the membrane.

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Yes, the GFP (D5.1) Rabbit mAb #2956 will detect Emerald GFP.

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We have found 100,000 cells per assay to be sufficient for enriching target loci for multiple histone modifications, transcription factors, and transcription cofactors. However, one can increase the number of cells to 250,000 per assay without scaling up beads, antibodies, enzyme, or buffers.

Learning Lab LIVE! Q&A with IHC Expert Jessica Simendinger

Antibody optimization in pilot experiments is recommended prior to running full-scale immunohistochemistry assays.