网页2015年3月24日 — In-gel Digestion Protocol for Mass Spec. By Ermelinda Damko, March 2008. Materials. Stained polyacrylamide gel containing protein(s) of interest. 50 mM ammonium bicarbonate (NH4HCO3; 3.96 mg/mL)/50% (v/v) acetonitrile. 10 mM dithiothreitol (DTT, 1.54 mg/mL) in 50 mM NH4HCO3. 55 mM iodoacetamide (10.2 mg/mL) in 50 mM NH4HCO3. 0.1% TFA.
网页In-Gel Tryptic Digestion Protocol. General Reagents. 100 mM Ammonium Bicarbonate: Dissolve .79g in 100mL MilliQ water. Place in a tightly capped bottle. Replace after 1 month. Acetonitrile. 45 mM Dithiothreitol (DTT 154.2g/mol): Make solution in a 1.5mL tube by weighing less than 10 mg DTT and adding appropriate amount of MilliQ water.
In-gel digestion for mass spectrometric characterization of …
网页2007年1月25日 — In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to ...
Updates of the In-Gel Digestion Method for Protein Analysis by …
网页Updates of the in-gel digestion protocol has been presented in the study. It has been shown that alternative reducing, alkylating agent reactions, and tryptic digestion buffers increase peptide and protein identification and reduce incubation times.
网页“In‐Gel” Digestion of Proteins in SDS‐Page Gel Slices for Mass Spec Protocol revised by Lori, June 2011 We recommend Gelcode Blue® Coomassie stain (Pierce) for detecting bands. This technique works for any band that can be seen by this stain. 0.1 to 0.2 micrograms of protein is ideal. Use a
网页2015年5月4日 — Digest overnight at 37 oC (or at least for 4 h). Centrifuge gel pieces (4min) and collect (keep) supernatant. Further extract peptides by one change of H2O and three changes of 5% formic acid in 50% CH3CN, incubate 20 min for each of the changes, centrifuge then collect at room temp.
In-Gel digestion - University of Washington
网页Use the In-Gel Tryptic Digestion Kit from Pierce, ThermoFisher or follow the protocol below. Here is a YouTube video preparing protein samples from SDS-polyacrylamide gels for mass spectrometry provided by the Proteomics core facility of Shared Resources, Fred Hutchinson Cancer Research Center.
Tryptic Digestion of In-Gel Proteins for Mass Spectrometry Analysis
网页2009年1月1日 — This chapter describes a tryptic digestion method for digestion of proteins in one-dimensional (1DE) or two-dimensional (2DE) polyacrylamide gels. The method involves cutting target protein bands or spots, removal of protein stain, reduction and alkylation of native protein, digestion and finally extraction of peptides for mass spectrometry ...
Protease Digestion for Mass Spectrometry | Protein Digest Protocols
网页A collection of protocols for in-gel and in-solution digestion with trypsin, trypsin/lysc or alternative proteases. Links to in-depth protocols and additional mass spec resources.
网页The Pierce™ In-Gel Tryptic Digestion Kit provides a complete set of reagents to perform approximately 150 digestions on colloidal coomassie or fluorescent dye-stained protein bands. The kit includes Thermo Scientific ™ Pierce Trypsin Protease, MS Grade, destaining buffers, digestion buffers, reduction reagents, and alkylation reagents. The
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