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It is possible to see a doublet at ~100 kDa for Grp94 due to post-translational modifications. Here is a link to our PhosphoSitePlus database detailing the possible post-translational modifications for Grp94: https://www.phosphosite.org/proteinAction.action?id=6928&showAllSites=true.
PTMScan® Sumoylation Remnant Motif Kit - SUMOylation Proteomics
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.
Additional internal testing has shown that the Caspase-3 (8G10) Rabbit mAb #9665 recognizes both Caspase-3 and Caspase-7. When this antibody was released to the market, we were unaware of the homology between the two proteins. We sincerely apologize for any inconvenience this may have caused.
Illustration of Motif Antibody Coverage
The YAP/TAZ (D24E4) Rabbit mAb #8418 antibody is generated using the TAZ human protein sequence (UniProt ID - Q9GZV5). However, the YAP sequence (UniProt ID - P46937) in the same region is only approximately 77% identical to the TAZ sequence used, which may affect how well the antibody binds to YAP.
KinomeView® Profiling Kit and Services
Crystals may be present in our Cell Lysis Buffer (10X) #9803 upon arrival due to the high concentrations of reagents included at the supplied 10X formulation, even when warmed to room temperature. This is not a cause for concern as your buffer is still good to use. We recommend warming the buffer to 37C for 5-10 minutes with mixing to help solubilize the crystals. Alternatively, diluting the 10X solution with ddH2O to at least 5X will yield a crystal-free solution. Once diluted, it can be aliquoted and stored at -20C for future use.
We have several antibodies for cleaved PARP that are validated for IHC-P in human samples. These are Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb #5625 and Cleaved-PARP (Asp214) (E2T4K) Mouse mAb #32563. Both work well and there is no preference for this application.
This video introduces PTMScan technology, a platform for the enrichment of post-translational modifications (PTMs) in mass spectrometry-based proteomics studies.
Webinar on mass spectometry used to investigate intracellular signaling activities via antibody enrichment of post-translational modifications
The IFN-α (6B18) Mouse mAb #3110 and IFN-α (8C21) Mouse mAb #3115 antibodies are different clones to the same target. These antibodies are approved for use with recombinant protein and will not detect endogenous levels of IFN-α. While either antibody will work well, #3110 is slightly stronger than #3115 by western blot and in addition has some citations in CiteAb.
Our Phospho-Akt (Thr308) (C31E5E) Rabbit mAb #2965 detects over-expressed levels of PKCalpha and beta. It also detects endogenous levels of PKC in Jurkat + CalA extract and possibly weakly in other endogenous models tested. Our Phospho-Akt (Thr308) (L32A4) Mouse mAb #5106 and Phospho-Akt (Thr308) Antibody #9275 detect over-expressed levels of PKCalpha and beta, but do not detect endogenous PKC. Our Phospho-Akt (Thr308) (244F9) Rabbit mAb #4056 detects over-expressed levels of PKCalpha and beta and may also weakly detect endogenous PKC in Jurkat + CalA extract, but not in other endogenous models tested. Our Phospho-Akt (Thr308) (D25E6) XP Rabbit mAb #13038 does not detect over-expressed PKC and is the best choice of the Phospho-Akt (Thr308) antibodies if this is a concern.
These antibodies target a post-translational modification (PTM) on DNA rather than protein. In order to adequately expose the epitope for binding, the DNA needs to be partially denatured. To accomplish this, we use ethanol and HCl. Traditional formaldehyde fixation followed by detergent-based permeabilization methods will not work for these PTMs.
Multimers are likely to demonstrate higher apparent affinity due to avidity effects. However, an exact recommendation for a competition assay would depend on other assay parameters, specifically assay format (in-tandem, classical sandwich, or premix), tags present on other proteins being used, and detection method.If you want to immobilize ACE2, we recommend using the Human ACE2 (18-652) Recombinant Protein (mFc-Tag) #83986 due to the ease of immobilization by protein A or anti-Fc. If you want ACE2 in solution, we recommend using the Human ACE2 (multimeric) (18-652) Recombinant Protein #85054 if you are immobilizing other proteins by protein A or anti-Fc. This is because the multimeric protein does not cross-react at protein A or anti-Fc surfaces. If you would like ACE2 in solution but are not using protein A or anti-Fc surfaces for immobilization, either protein should work well. Finally, if you want the most potential avidity in solution, then the multimeric protein is preferable.
To avoid intra-sample batch effects during liquid chromatography with tandem mass spectrometry (LC-MS/MS), we recommend a run order that involves running one technical replicate of each enrichment, then the remaining replicates in reverse order. For example, samples 1-4 have two replicate injections each: 1A/1B, 2A/2B…, etc. Therefore, the run order would be: 1A, 2A, 3A, 4A, 4B, 4A, 3B, 2B, 2C, 1C.
After the final purification step, we typically observe NGS libraries with a concentration ranging between 10-30 ng/ul. If the library DNA yield is too high, less PCR cycles can be considered.
Comparison tables for product 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.
Watch this video to learn how two different methods (antibody-based immunoaffinty enrichment and IMAC) combine for improved coverage of the phosphoproteome.
This video presents tips to help your immunoprecipitation experiment: how to avoid IgG masking; optimizing epitope tags; experimental design (controls); what to consider when choosing lysis buffers, antibodies, and beads.
This video describes the simplified PTMScan® HS workflow for LC-MS analysis of ubiquitination and SUMOylation that improves readout sensitivity, reduces sample prep time, and eliminates lyophilization, 9M urea, and antibody elution steps.
SignalStar™ Multiplex IHC Kits & Reagents have not yet been validated for use in frozen tissues. We are currently in the process of validating our antibodies and protocols for use in fresh or frozen tissue.
What to know about widefield versus confocal imaging settings for your microscope to get optimal immunofluorescence data.
The concentration of the pAG-MNase Enzyme #57813 in our CUT&RUN kits is 25ug/ml.
The GFP Antibody #2555, GFP (4B10) Mouse mAb #2955, GFP (5G4) Mouse mAb #55494, and GFP (D5.1) XP® Rabbit mAb #2956 are not expected to detect Turbo GFP due to low sequence homology.
Learn about the power and applications of recombinant antibodies in research. Click here to find out more and revolutionize your experiments.
It should not matter whether you spike in IgG2a or IgG1 antibodies when using the SARS-CoV-2 Spike RBD-ACE2 Blocking Antibody Detection ELISA Kit #69486, as the kit is measuring the ability of the sample to block the ACE2-RBD interaction and is not directly measuring the antibodies. Specifically, this kit is designed to detect blocking antibodies present in human serum or plasma samples.
Caspase Cleavage Substrate Proteomics
Whether you're learning a new technique for the first time, or need to troubleshoot problems in your experiments, CST scientists are here to provide guidance. The same application experts that develop and validate our antibodies are here to share their k