Search Methods. rinsed with 70% ACN/0.1% formic acid before use. Excess ion-pairing reagent may cause retention loss due to various electrostatic effects associated with adsorption of the ion pairing reagent on the silica surface. (2001). 6. Store any remaining Lys-C solution Features of the Mass Spec Sample Prep Kit: Learn more about the Thermo Scientific Pierce Mass Spec Sample Prep Kit. or gel filtration (desalting columns). The preparation of special buffer solutions is described in the sections in which their use is specified as in the microbiological assay of antibiotics or in the individual monographs where the use of such solutions is indicated. salts, enzymes, inhibitors, detergents, denaturing/chaotropic agents, reducing/alkylating/peptide Any thoughts? When using 10g of cell lysate, in blood plasma). Standard (Product No. slices. TFA may also form strong hydrophobic interactions with silica based HPLC columns, substantively altering the column chemistry. Add 100l of Cell Lysis Buffer to the tube andgently of CellLysis Buffer for a 20l cell pellet). Add 75 L Digestion Solution (enzyme-to-protein ratio 10. These reagents also linger for much shorter times within ESI sources. digestions for protein identifications in proteome studies. Repeat thisstep twice.5. 2. Mass Spectrometry Sample Preparation Procedure for Protein Samples Well, this procedure is good enough for a rough screening solution- call it a quick and dirty method. Discard any unused DTT solution.6. FASP Protein Digestion Kit, Expedeon P/N 44250, Thermo Fisher P/N EX44250Kit Contents (sufficient for processing 8 samples): 1. Buffer Considerations for LC and LC-MS - Chromatography Online 23225) or Thermo Scientific Pierce BCA Protein Assay Kit-Reducing Agent Compatible (Part No. In initial studies we optimized the conditions for trypsin digestion of cellular lysate proteins with LC-MS/MS analysis on Thermo Scientific Velos Pro and Thermo Scientific Orbitrap XL instruments. Prepare Reducing Buffer as described in the Material Preparation Section. A One-Step Solid Phase Extraction Method for Bioanalysis of a The final concentration of TCEP in the . Mix up to 30 L (0.4 mg) of a protein extract with 200 L of 1. 11, 11201130 (1997), 8. Use a spot picker or scalpel to excise protein band of interest from 1D or 2D gel. Add 11.5l of IAA solution to the sample (final IAA concentration is ~50mM). samplevolume to 100L using Cell Lysis Buffer to a final concentration of 1mg/ml. Add 50l 0.5 M Sodium Chloride Solution provided with the FASP Kit and centrifuge Mass Spectral Effects from Using Ammonium Bicarbonate for Protein IEX
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