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We recommend dilution of the primary antibody into PBS containing 1% BSA and 0.3% Triton X-100. BSA acts as a carrier protein and improves antibody stability in dilute environments while Triton X-100 aids in ensuring maximum cell coverage. We recommend overnight incubation to allow maximum time for antibody binding. If the incubation time needs to be shortened, we generally recommend at least 2 hours at 37C but performance cannot be guaranteed.
We have tested the reactivity of the Phospho-β-Catenin (Ser33/37) Antibody #2009 to Ser33/37 by peptide dot blot. This antibody showed strong signal when phosphorylated at Ser37 alone and when phosphorylated at both Ser33 and Ser37. There was weak signal when the peptide was phosphorylated at Ser33 alone, suggesting that the Ser37 contributes more strongly to antibody binding compared to Ser33. No signal was seen when these sites were not phosphorylated. It is our understanding that in vivo the sites (S33/S37/T41) in this region are progressively phosphorylated as a cassette so individual phosphorylated sites would likely be rare in vivo.
A scientific resource for the ENTH protein domain containing information on structure, function, and domain binding to phospholipids during endocytosis.
We use whole cell extracts when validating and QC testing our histone antibodies by western blot and do not routinely test purified histones. We generally load 20-30ug of whole cell extracts on the gel but have seen good signal with less than 20ug for some of our stronger histone antibodies. Since we do not routinely test purified histones we do not have a specific amount we can recommend. We suggest titrating your purified histones to determine the optimal amount to use for your specific antibody. Most of our histone antibodies are very strong, but this should be empirically determined for your specific samples.
A scientific resource for the EVH1 protein domain containing information on structure, function, and binding to a proline-rich motif to regulate actin dynamics.
Xenophagy provides an important defense against foreign pathogens such as bacteria and viruses by targeting them for degradation through autophagy.
A multiple antibody strategy is a powerful approach to antibody validation. This provides confidence that antibodies are binding the correct biomolecule.
Targeting B-Cell Maturation Antigen Holds Promise for Multiple Myeloma Cure. B Cells, TNFR, CAR-T, hematopoietic, bone marrow, BCMA, ADCs, Multiple myeloma
CST Science Scholarships consists $10k scholarships, awarded to high school juniors, from Massachusetts, to pursue a degree in a scientific discipline.
Activation of signaling networks downstream of B-cell receptors leads to altered B-cell phenotypes, which can be identified by cellular markers.
KinomeView Protocol: easy to follow directions describing the step by step experimental procedure.
All four of our MMP-2 antibodies should detect both the full-length proenzyme that runs at 72 kDa and the cleaved, active enzyme that runs at 64 kDa. As the activated form is secreted, it is more difficult to detect using endogenous models and often the 72 kDa proenzyme is more easily detected. In very high expression cell lines, such as U-87 MG, SF-296, and 3T3, the active form can be detected as a faint signal.
Unfortunately, we cannot confirm that the recombinant SARS-CoV-2 spike proteins we provide are in exactly the same conformation that would be expected in nature, as we do not yet have direct data addressing this issue. That being said, our recombinant SARS-CoV-2 spike proteins are expressed in mammalian cells to try to ensure the presence of secondary (post-translational) modifications, which would not happen in a prokaryotic expression system. Furthermore, we suspect the trimer is closer to native folding than the monomer, but without NMR or X-Ray crystallography, we have not been able to confirm this.
We have tested a representative group of rabbit polyclonal, rabbit monoclonal, and mouse monoclonal antibodies and saw no change in their activity after two freeze/thaw cycles on dry ice (-78.5C). However, because freeze/thaw cycles are known to cause damage to antibodies, we cannot offer any firm guarantees.Most of our off-the-shelf antibodies are supplied in a 50% glycerol buffer and will not freeze when stored as recommended at -20C. Regrettably, we do not have sufficient stability data for long-term storage at other temperatures. Therefore, for ideal long-term storage, antibodies should be stored at -20C. Any potential damage from repeated freeze/thaw cycles is avoided at this temperature.
Immunoprecipitation Protocol (For Analysis By Western Immunoblotting) : easy to follow directions describing the step by step experimental procedure.
We observe multiple bands at or around 33-35 kDa in cells with high STING protein expression, such as THP-1 and HDLM-2. Regrettably, we are not certain about what the lower molecular weight band represents in the THP-1 extract. However, customers may be interested in the following possibilities:1) Please see Wang, P. et al. (2018) Nucleic Acids Res. 46, 4054-4071 (PMID: 29547894; https://pubmed.ncbi.nlm.nih.gov/29547894/). The two bands in THP-1 extract may represent the alpha and beta isoforms of STING.2) Please see Rodríguez-García, E. et al. (2018) Immunohorizons 2, 363-376 (PMID: 31026807; https://pubmed.ncbi.nlm.nih.gov/31026807/). STING isoforms 2 and 3 do not include #13647’s epitope. However, STING isoform 1 may be detected by #13647.3) Given that the lower band is also observed in 293T cells transfected with a construct expressing human STING protein (see image on the product webpage; https://www.cellsignal.com//products/primary-antibodies/sting-d2p…
Immunoprecipitation of Denatured Proteins Protocol: easy to follow directions describing the step by step experimental procedure.
Webinar: Achieving Reproducibility: Don't Let Antibodies Be a Variable in Your Experiment
Immunoprecipitation Protocol (For Analysis By Kinase Assay): easy to follow directions describing the step by step experimental procedure.
Cell Signaling Technology (CST) complies with and is subject to the EU-U.S. Privacy Shield Framework and the Swiss-U.S. Privacy Shield Framework as set forth by the U.S. Department of Commerce regarding the collection, use, and retention of personal info
Immunofluorescence Protocol without Permeabilization: easy to follow directions describing the step by step experimental procedure.
In-Cell Western Protocol: easy to follow directions describing the step by step experimental procedure.
While performing the BrdU cell proliferation assay, we also notice some changes in cell morphology after adding the fixing/denaturing solution. However, in our experience, it does not adversely affect the signal obtained with the kit. It should be noted that we do not recommend any washes after fixation/denaturation. Instead, we recommend waiting until after incubating the detection solution for one hour at room temperature. No centrifugation step is required unless starting with suspension cell lines.
For years, the TME could only be viewed in single-cell snapshots by flow cytometry or limited tissue staining. IMC offers a panorama of this dynamic world.
To help you pick the best reagents for your assay we will spend the next several posts reviewing how companion reagents affect IHC.
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New antibodies for Alzheimer's research were presented at SfN 2022, including tools to study neuroinflammation in glial cells like microglia and astrocytes
Unfortunately, we have not internally validated the use of acidic samples with our Cyclic AMP XP(R) Assay Kit #4339. We have had customers try this method, preparing their samples with 0.1M HCL and then neutralizing. The customers did not have success with this method.If using acidic sample preparation, we recommend further diluting samples in 1X PBS after neutralization, prior to adding samples to the plate. Again, as we have not internally validated this, we cannot guarantee this kit will work with acidic samples.
A journal club article elucidating the mechanisms of beta cell failure leading to type 2 diabetes.
Immunohistochemistry (IHC) is a powerful technique. Learn how to perform IHC on paraffin sections with our step-by-step IHC protocol. Click here.