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We currently have four different secondary antibodies approved for use in fluorescent western blotting, depending on species and wavelength of detection:
Learn about the history of Cell Signaling Technology and our commitment to providing reproducible tools to move your science forward.
Products and Related Resources for Cytokines and Inflammation SARS-CoV-2 Research
The transcriptional activity of c-Jun is regulated by SAPK/JNK phosphorylation at Ser63 and Ser73. The signaling pathway can be found at https://www.cellsignal.com/contents/science-pathway-research-mapk-signaling/sapk-jnk-signaling-pathway/pathways-mapk-sapk. Cellular stressors, such as UV radiation, inflammatory cytokines, or ceramide, activate Rho-family GTPases and subsequent MAPKKK (Map Kinase Kinase Kinase) pathways. MAPKKKs can activate MKK4/7, which directly phosphorylate and activate SAPK/JNK. Upon translocation to the nucleus, SAPK/JNK can activate c-Jun and other transcription factors. Proteins generally undergo a phosphorylation dependent mobility shift (PDMS) in SDS-PAGE. This shift can vary from target to target, and even with different activation events on the same target. Recent experiments suggest this is due to the additional negative charge conferred by phosphorylation sites disrupting protein binding to SDS (see https://www.researchgate.net/publication/264031559_Phosphorylation-Depe…
The Pan-Cadherin Antibody #4068 can detect R-, N-, E-, and VE-Cadherin. It does not detect P- or K-Cadherin. Reactivity to other Cadherins has not been tested.
The larger NRF2 size is likely due to a secondary structure or modification, which is not uncommon. Under normal conditions, KEAP1 targets NRF2 for degradation through the polyubiquitination/proteasome pathway, which makes it hard to detect the protein in cells (some cell lines have mutations in the NRF2 pathway that may cause NRF2 accumulation). After certain treatments, KEAP1 is modified and cannot bind NRF2, so NRF2 is not polyubiquitinated and will accumulate and enter the nucleus to activate transcription of its targets. Because polyubiquitination may result in different numbers of ubiquitin molecules binding to the same target protein, most polyubiquitinated proteins will not appear as distinct bands on western blot, but as a smear or ladder in some cases.
Drs. Schietinger and Youngblood review the underlying mechanisms and epigenetic programs determining tumor-specific T-cell dysfunction and reprogrammability and discuss current challenges and outstanding research questions in cancer immunotherapy.
Infiltrating myeloid cells are analyzed in the tumor microenvironment using multiplex IHC, employing new monoclonal antibodies to IDO1 and Arginase.
To help cancer researchers to push their field forward, CST scientists dig into the literature to identify unmet needs. Dr. Cigall Kadoch shares her thoughts on what makes CST antibody development and validation unique in this video.
The composition of our Lysis/Binding/Wash Buffer #11524 is as follows: 25 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM MgCl2, 1% NP-40 and 5% glycerol.
As the importance of the mammalian SWI/SNF (BAF) complexes in contributing to cancer became apparent, Dr. Kadoch realized there was a real lack of reagents to effectively study this class of proteins.
Cigall Kadoch PhD keeps a running list of targets to advance mechanistic studies of human disease. Here, Dr. Kadoch discusses the value of collaborating with CST to develop novel reagents to probe ATP-dependent chromatin remodeling complexes.
Products and Related Resources for Viral Entry, Replication, and Transcription SARS-CoV-2 Research
We suggest a sequencing depth of 3-5 million reads per sample. Because of the very low background signal generated in CUT&RUN, this sequencing depth is usually sufficient for histone modifications and transcription factors. We have found that less than 3 million does not work and greater than 15 million greatly increases the number of duplicate reads.
Even though EGR1 has a predicted size of ~58kDa, 75kDa is the correct running size. This is because EGR1 is Sumoylated, which along with other PTMs, increases the size it runs at on a western blot.
We have not back calculated the number of viable cells per well based on the OD readout from the XTT Cell Viability Kit #9095. We use the OD readout to see the activity level of the wells. We have been unable to find any calculations that could be of use for determining the number of viable cells from the OD readout. Unfortunately, we do not have any additional information to provide.
Our Anti-biotin, HRP-linked Antibody #7075 is intended for use with our Biotinylated Protein Ladder Detection Pack #7727. However, it is possible for this antibody to also detect endogenously biotinylated proteins in cell lysates if present in sufficient quantities. If this type of interaction is suspected in a given experiment, it is recommended that the Biotinylated Protein Ladder be probed separately from the cell lysate lanes to avoid detection of endogenously biotinylated proteins in the experimental samples.
Hallmarks of Antibody Validation-Complementary Strategies that Employ Western Blot, Immunohistochemistry (IHC), Immunofluorescence (IF/ICC), Flow Cytometry, Histone Antibody, ChIP-qPCR, ChIP-seq.
Products and Related Resources for Type I Interferon and Viral Restriction Factors SARS-CoV-2 Research
When using the Senescence β-Galactosidase Staining Kit #9860, we regularly scale up and down the amount of buffer used. If you are working with a 96 well plate, we recommend adding more buffer than 50ul per well. As long as the staining solution is made with the same concentration of reagents, adding more buffer should not give you a false positive or negative (assuming the pH of the final staining solution is within the acceptable range of 5.9-6.1). A volume between 200-250uL should be appropriate for a 96 well plate format.
A multiple-antibody strategy is a powerful approach to antibody validation. The most common method to achieve this is to immunoprecipitate (IP) the target with one antibody and subsequently detect it by western blotting with another antibody against the
Our continuing goal is to make the highest quality, most consistent antibodies that work the first time, every time.
Learn how RNA-binding proteins influence RNA regulation and epitranscriptomics and contribute to the progression of a variety of diseases.
Content Overview for SARS-COVID Research
KinomeView Profiling is a western blot based screening technique used in conjunction with motif antibodies to optimize conditions (time, dose, cell line, compound) for maximum differential response. This can then be used to configure a follow-up PTMScan study with the appropriate motif antibodies.
CDCP1 is cleaved between Arg368 and Lys369 resulting in a C-terminal fragment that migrates at approximately 70 kDa.
Watch this webinar for applications, tips, and tricks for Simple Western, for fast, reliable, and hands-free automated western blotting using CST antibodies. See an overview of the CST-Bio-Techne partership, brining westerns into the 21st century.
The solution formulation of the PTMScan Control peptides is 25% acetonitrile, 74.9% water, 0.1% trifluoroacetic acid (V:V).