IP Profiling and Pathway Analysis

IP Profiling & Signaling Pathway Analysis

This is a very popular service for molecular biologists who are curious about the binding parteners of their proteins of interest. When you are studying the function of a gene product (such as a protein) or its cellular location, you will always need to answer the question of what other proteins this protein binds to, what signaling pathways this gene is involved. The Omics Technologies IP Profiling Service is designed to provide the most complete and accurate information about the full catalogue of proteins present in your co-immunoprecipitated protein complex containing your protein(s) of interest. Based on our list of your proteins' essential partners, you may identify a promising novel signaling pathway or identify your protein of interest as a novel part of a well known signaling pathway. All of these findings which will be an critical part of your molecular biology project.

We will provide you for free our patent-pending My-IPElu™ buffers and protocols to help you prepare your (co-)immunoprecipitation samples when you order the service with us. You will be asked to provide us your IP'd or Co-IP'd protein complex together with your control pretreated with our My-IPElu™ buffers. Your samples will be digested with Trypsin and peptides are analyzed by world's most advanced Orbitrap LC-MS/MS. Fingerprints of your mysterious binding parteners are generated and matched to protein sequence database. Report will be sent to you in Excel format as well as a summary in PDF format. We will also provide you any details you need for your papers' MATERIALS AND METHODS section. We will make sure you understand your result and help you with your paper writing with free follow-up services.

1) IP sample harvest – Immunoprecipitated protein complex samples are harvested through elution using My-IPElu™ buffers.

2) Sample preparation – Cell or tissue lysis, biofluid (plasma or serum) depletion; protein quantification on all samples.

3) Sample digestion – Reduction and alkylation followed by in-solution trypsin digestion and C18 cleaning before labeling.

3) Sample labeling – Isotope labeled reagents will be applied to label 2 through 10 samples individually simultaneously.

4) SCX cleaning – Ion-exchange to remove excessive chemicals and fractionation of pooled sample through off-line SCX HPLC.

5) 2D fractionation – bRPLC separation of the SCX cleaned samples into 96 fractions, and further pooled into 24 fractions according to MyProt™-iPooling algorithm (based on peptide hydrophobicity, IEP, etc.)

6) Nano LC/MS/MS – 50 nL/min liquid chromatography to enable super high ionization efficiency for downstream Mass Spectrometry Analysis using the state-of-the-art Orbitrap Mass Spectrometer. 24 fractions will be analyzed in 24 Mass Spectrometry runs.

7) Protein profiling – Protein database searching with Mascot, Sequest HT, PEAKS de novo approaches, etc.

8) Data analysis – Data validation, visualization and quantification using commercial softwares and Omics Technologies's unique R packages and scripts.

9) Pathway analysis – Molecular Biology Signaling Pathway Analysis through commercial softwares and Omics Technologies's in-house developed statistical algorithms.

10) Report generation – Report will be sent to you in Excel format as well as a summary in PDF format. We will also provide you any details you need for your papers' MATERIALS AND METHODS section. We will make sure you understand your result and help you with your paper writing with free follow-up services.

1, IP'd or Co-IP'd protein complex (pretreated with My-IPElu™ buffers)

2, Customized sample types (please contact us to discuss)

Please refer to our Omics Technologies Proteomics Sample Preparation and Shipping Guidelines for instructions on how to prepare your samples with less contamination for Mass Spectrometry Analysis.