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The FastScan kits differ from the PathScan kits in that (1) they come with a positive control and (2) the assays can be performed much faster. With FastScan, the samples are incubated with a capture antibody conjugated with a proprietary tag and a detection antibody linked to HRP with no need for wash steps. The FastScan kits require the use of a specific lysis buffer, included in the kits, and are not compatible with other buffers. In terms of sensitivity, FastScan and PathScan are fairly equivalent.

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Human samples will have 55 kDa and 25 kDa bands, representing the long and short isoforms (FLIP-L, FLIP-S). A third isoform, FLIP-R, was originally identified in Raji cells and migrates at approximately 25 kDa [see Djerbi, M. et al. (2001) Scand. J Immunol. 54, 180-9 (PMID: 11439165; https://www.ncbi.nlm.nih.gov/pubmed/1439165) and Golks A. et al. (2005) J. Biol. Chem. 280, 14507-13 (PMID: 15701649; https://www.ncbi.nlm.nih.gov/pubmed/15701649)]. Mouse FLIP is alternatively spliced, where only the long isoform is generated and, in some rare cases, the FLIP-R isoform may be observed. Frequently, the shorter isoforms undergo proteasomal degradation in mice and are not detected by western blot.

 

DNA Library Prep for CUT&RUN: A Visual Guide

A comprehensive, step-by-step video of the CUT&RUN DNA Library Prep Protocol for Illumina systems, picking up after the CUT&RUN protocol video. Key protocol steps and tips are highlighted.

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We have observed >1E6 peak area for each control peptide when used at the supplied concentration followed by PTMScan and PTMScan HS enrichment, as detected by our LCMS instruments.  However, the limit of detection (LOD) depends on many factors such as the sensitivity of the instrument and the LC gradient that is unique to each user.  Users may adjust the concentration of the supplied peptides as needed to find an appropriate signal strength that has a sufficiently high signal-to-noise ratio for reliable detection.
 

Neurodegeneration Drug Development

Streamline your neurodegeneration therapeutic development with CST recombinant monoclonal antibodies, ELISA and cellular assay kits, custom products, and services.

Stress Granule Life Cycle Diagram

Necrotic Cell Death

Necrosis has been classically defined as an unprogrammed form of cell death that occurs in response to overwhelming chemical or physical insult.

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The antigen for our Phospho-PI3 Kinase p85 (Tyr458)/p55 (Tyr199) Antibody #4228 shares 100% sequence homology with human. However, we have tested many different human samples in-house and have not been able to obtain signal with this antibody.

Multiplex IHC

Multiplex IHC - Being able to visualize multiple targets simultaneously is important in research fields like immunology & oncology. Learn more here.

Glutamine Metabolism

Expert-reviewed interactive pathway providing a current overview of Glutamine Metabolism signaling pathway.

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PTMScan® is compatible with the use of stable isotope labeling using amino acids in cell culture, or SILAC. The incorporation of heavy stable isotopes has no effect on antibody-peptide binding. The key point in employing SILAC is to grow the cells in the presence of the heavy amino acids for five to six generations to ensure an incorporation efficiency of 97% or more for accurate qualitative and quantitative results.
 

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Tissue samples should be placed in 1 ml of urea lysis buffer per 100 mg of tissue wet weight. For example, a 300mg tissue sample would be homogenized in 3mls of urea lysis buffer. We use a Polytron tissue homogenizer, and homogenize the sample 3 times for 10 seconds, then continue the protocol at section 1, B. 3. and sonicate the homogenate before centrifugation. Please note that the acetyl-lysine antibody will cross react with carbamylated lysine. To avoid this, please make sure that the urea lysis buffer is made fresh, and perform the reduction with DTT at room temperature to reduce any carbamylation.

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The β-Actin (8H10D10) Mouse mAb #3700 has kappa light chains.

Spatial Imaging as a Tool for Discovery of New Candidates to Better Predict Response to Immunotherapies

Dr. David Rimm of Yale School of Medicine discusses multiplexing spatial imaging technologies and their development to improve response prediction for immunotherapy. This webinar was recorded at AACR 2022.

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We have several antibodies for ribosomal protein S6 (rpS6) that are validated for IHC-P with mouse samples. These are S6 Ribosomal Protein (5G10) Rabbit mAb #2217 and S6 Ribosomal Protein (54D2) Mouse mAb #2317. For this application, S6 Ribosomal Protein (5G10) Rabbit mAb #2217 is preferred, as S6 Ribosomal Protein (54D2) Mouse mAb #2317 could be problematic due to mouse on mouse (MOM) background staining.

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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 Concanavalin A beads used for cell binding, or when you hope to freeze the cell pellets for future use. If fixation is necessary, refer to the CUT&Tag Assay Kit Protocol, Appendix A to lightly fix cells prior to the CUT&Tag experiment. We find that live cells produce better results than fixed cells in the CUT&Tag assay. Therefore, we recommend using live cells, if possible, unless fixation is necessary for the scenarios mentioned above.

Rheumatoid Arthritis Pathogenesis Signaling Pathway

Expert-reviewed interactive diagram of Rheumatoid Arthritis Pathogenesis and relevant antibodies for studying targets associated with the autoimmune disease.

Microglia in Neurodegeneration

Contribution of Extracellular Matrix to EMT

Expert-reviewed interactive pathway providing a current overview of Contribution of Extracellular Matrix to EMT Signaling.

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Our MT1-MMP (D1E4) Rabbit mAb #13130 was produced with an antigenic peptide corresponding to residues surrounding Met293 of human MT1-MMP protein (see UniProt ID #P50281; https://www.uniprot.org/uniprot/P50281). Therefore, the antibody recognizes both the full-length MT1-MMP zymogen and the proteolytically processed, active enzyme (residues 112 to 582). The 62 kDa band represents the inactive zymogen that includes the inhibitory prodomain. MT1-MMP is activated by cleavage between Arg111 and Tyr112 to produce a mature/active enzyme [see Golubkov V.S. et al. (2007) J Biol Chem 282, 36283-91 (PMID: 17938169; https://www.ncbi.nlm.nih.gov/pubmed/17938169)] that migrates at approximately 50 kDa. We have not performed any direct testing to confirm that the 50 kDa band is definitively the cleaved form, but it is very likely the case based on the literature, expression levels, and epitope location.

Simplified Staining: Signaling Analysis in Heterogeneous Populations by Flow Cytometry

Watch this 20-minute webinar for tips and tools to optimize simultaneous incubation of different antibodies, plus insignts into maximing multiplexing efficiency while maintaining assay quality.

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We recommend washing the Protein A and Protein G magnetic beads prior to use to help clear them of any potential increase in background due to the buffer in which they are provided. 

Contribution of Soluble Factors to EMT

A number of growth factors and cytokines can induce an Epithelial-Mesenchymal Transition (EMT) in tumor cells.

Tumor Angiogenesis

Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, plays a key role in tumorigenesis.

Listen to Your Gut: Deciphering the Role of Immune Cells in Inflammatory Gut Disorders

Drs. Cominelli and Kugathasan describe new techniques to identify and target inflammatory signaling in gastrointestinal immune cells in this webinar.

TMT Labeling and Detection

TMT Labeling and Detection

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We typically perform this assay using the Senescence β-Galactosidase Staining Kit (#9860) from start to finish in one day and have not paused between fixing and staining. Therefore, we cannot guarantee the results will not be negatively impacted if cells are incubated in PBS buffer after fixing instead of immediately proceeding to overnight staining. However, we have spoken with numerous customers who have paused after fixing, stored their cells in PBS at 4C for a few days prior to staining and have had great success.

Modulating Signaling Activities with Activators & Inhibitors for Western Blot Experiments

How to combine modulators with selection of antibody targets and controls in western blot experiments.