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Resources results for "ptmscan hs"

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Reproducibility in Proteomics Experiments: Using Control Peptides with PTMScan

Control peptides can be used to ensure reproducibility in proteomics experiments conducted with PTMScan.

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Through in-house testing, we have found that the magnetic beads from our PTMScan HS kits aggregate and stick to the walls of some specific brands of tubes. Moreover, they cannot be washed off or spun off the sides of the tubes. This is an issue with specific brands of tubes themselves and not an inherent issue with the PTMScan bead product. Therefore, the following brands of polypropylene tubes are NOT recommended with magnetic bead PTMscan HS kits: 
1. CellTreat #229441, polypropylene 1.5mL tube, non-sterile, non-pyrogenic
2. Denville Scientific (Harvard Bioscience) #C3171, 1.5mL Posi-Lock tubes, polypropylene

The following brand of microcentrifuge tubes is compatible with magnetic bead PTMscan HS kits:
1. USA scientific tubes #1615-5500
 

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The PTMScan HS Magnetic Immunoaffinity Beads should not be frozen. We recommend storing this product at 4C. However, we have performed functional testing by IAP and subsequent mass spec analysis after 1X round of freeze thaws and found that the beads performed as expected. If the beads are accidentally frozen and thawed once, they should be fine to use, but we first recommend submitting a technical support inquiry to discuss this with a CST product scientist.

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We recommend using 10 mg of protein per immunoaffinity purification (IAP) sample for our standard PTMScan kits. For the PTMScan HS product line, 1 mg of starting material is optimal. The minimum sample amount is dependent upon the cell line/tissue and the endogenous levels of post-translational modifications (PTMs). If less protein is used, fewer identifications could be expected.

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The PTMScan® HS Ubiquitin/SUMO Remnant Motif (K-ε-GG) Kit #59322 uses the same antibody as in the traditional PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit #5562 and PTMScan Pilot Ubiquitin Remnant Motif (K-ε-GG) Kit #14482. This means it has the same affinity for the K-e-GG remnant after trypsin cleavage.

However, we have changed the antibody – bead linkage to be near-covalent in strength so the antibody does not contaminate the PTM peptides during the elution step. This prevents issues with the liquid chromatography column clogging and increases the yield of PTM peptides identified. The specificity is also improved with changes to the bind and wash buffers that help reduce non-specific peptide binding.

In addition, the new HS kit beads are magnetic, rather than agarose, which simplifies the workflow by eliminating multiple centrifugation steps during the bead washing and elution steps.

 

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The “classic” PTMScan® kits that use agarose beads and the PTMScan HS kits that use magnetic beads are both designed for a single PTM peptide enrichment. The nature of the acidic elution step, which leads to the release of bound PTM peptides, denatures the antibodies. This denaturing step alters the conformation of the antibodies. Therefore, employing these antibodies for subsequent enrichments may produce inconsistent results in PTM peptide enrichment.
 

Solutions for Targeted Protein Degradation

Streamline Targeted Protein Degradation Drug Discovery with specific, application-validated antibodies and industry-leading proteomic services.

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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.
 

A Revolution in Proteomics Analysis: PTMScan 20 Years Later

What if an antibody could identify a PTM, regardless of the flanking amino acid sequence? This innovative idea is the basis of CST's PTMScan product line.

Highly Sensitive Post-Translationally Modified Peptide Enrichment for LC-MS Analysis

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.

Japan Regulation/MSDS

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Antibodies and beads in the PTMScan® HS Ubiquitin/SUMO Remnant Motif (K-ε-GG) Kit #59322 are conjugated such that the antibody will not elute from the bead during peptide elution. The exact method of conjugation is proprietary.

PTMScan® Motif Antibody Kits

PTMScan Motif Antibody Kits from Cell Signaling Technology

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The background binding of non-PTM peptides is typically 50% or more of the total peptides bound to the antibody beads in our agarose PTMScan® kits. This has been improved to less than 50% background or non-target PTM peptides for the magnetic PTMScan® HS kits. Therefore, it is hard to say from sample to sample exactly how much binding is from the antibody and how much is from the background. 
It is important to use a high-capacity stage tip that can handle 10ug of peptide at the end of the protocol after the enrichment step. In addition, we usually run 33% of each enrichment 2X for technical replicates for each sample.

Product Shelf-Life

A table that lists shelf-life expectations for CST products (when stored under the recommended conditions).

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The incubation time for PTM peptide enrichment has been empirically determined with an optimum time of ~2 hours. Incubation times greater than 2 hours show no increase in PTM-specific enrichment, and shorter times may produce unwanted variability in the qualitative results.
 

Motif Antibodies for PTMScan® Discovery Services

PTMScan Motif Antibodies and Kits, for customers who purchase a license and would like to perform PTMScan Proteomics in-house.

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The methods for the LC and MS are shown below:
 

 ParameterSetting
Sample prepIP sample resuspension volume20 uL
 Sample volume per injection4 uL
 

Simplifying Proteomics - Part 2

Immunoaffinity purification of PTM peptides for mass spectrometry-based proteomics--PTMScan

PTMScan Tutorial Video

CST PTMScan tutorial video explains how to use antibodies to quickly and efficiently enrich PTM peptides for mass spectrometry-based proteomics

Cell Signaling Technology, Inc. Announces Renewal of PhosphoScan® Proteomics Research Project for Lead Compound Biomarker Discovery

Nov 2008, Renewal of a research agreement with Ortho-Biotech Oncology Research & Development, under which CST will employ its patented PhosphoScan® and PTMScan® technologies in research aimed at biomarker discovery and validation for multiple enzyme target classes and lead small-molecule inhibitors.

PTMScan® Pathway Service

Features, benefits, and product listing for PTMScan Direct Proteomic Services for the targeted screening of defined protein sites critical for drug and biomarker discovery.

Got the "NOS"? Fueling Research with New Nitro-Tyrosine Antibody Tools

To study pathways regulated by nitrotyrosine, CST offers two recombinant monoclonal antibodies against the same PTM for WB and proteomic applications.

KinomeView® Profiling

Information on KinomeView western blotting service offered by Cell Signaling Technology that uses Phospho-Motif Antibodies to provide a kinome-wide view of cellular phosphorylation.

Proteomics Kits

PTMScan® Discovery Proteomics Services

CST can identify and quantify thousands of post-translational modification sites specific to your sample—phosphorylation, ubiquitination, acetylation, and more.

LC/MS Proteomics Resource Center

The power of LC/MS proteomics Learn about PTMScan technology for proteomics from CST. Enrichment of PTM peptides combined with mass spec. Click

Webinar - Simplify Proteomics

Matthew Stokes, Ph.D., and Christopher Rose, Ph.D. introduce Cell Signaling Technology’s PTMScan® method and provide a demonstration of its applications.

PTMScan® Videos and Webinars

PTMScan videos and links to complementary technologies for phospho-peptide enrichment and Science Webinar on Proteomics and Cell Signaling.

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Yes, the flow through from a PTMScan experiment can be saved and stored at -80C for future enrichments. We have performed 5 different PTM enrichments sequentially from one sample. The limitation would be the choice of the initial protease used for the digest. For example, a UbiScan PTMScan requires peptides digested with trypsin and the flow through peptides may not be ideal to capture peptides that require an arginine (R) residue in the phospho-motif as in the AKT or AMPK substrate PTMScan kits.