Material Provided:
Each vial contains 150 μl of OxyBlot™ Protein Standard, which is sufficient for 60 minigels.
Instructions for Use:
Warm the mixture to room temperature. (Since the mixture contains SDS that may precipitate during storage, it may be necessary to heat the stock solution to 50°C to redissolve the SDS). Combine 2.5 μl of the OxyBlot™ Protein Standard with 20 μl of 1X Gel Loading Buffer prior to loading onto the minigel. Do not heat prior to gel loading. Perform SDS-PAGE according to standard procedures.
NOTE: Since the proteins in the standard mixture already contain DNP residues, the protein standard should not be subjected to the derivatization reaction in the OxyBlot™ Protein Oxidation Detection Kit protocol.
In order to maintain consistency, the 2-Mercaptoethanol must be omitted from the 1X Gel Loading Buffer if a reducing agent was not used in the preparation of the experimental samples.
Reagent Preparation:
1X Gel Loading Buffer
To make 8 ml:
0.5 M Tris-HCl, pH 6.8 1.0 ml
Glycerol 0.8 ml
14.3 M 2-Mercaptoethanol (Reagent Grade) 0.1 ml
10% SDS 1.6 ml
0.05% Bromophenol Blue 0.32 ml
dH2O 4.18 ml
Divide into 1 ml aliquots and store at -15°C to -25°C.
Protein Molecular Weight
Phosphorylase B 97,400
Bovine serum albumin 68,000
Ovalbumin 43,000
Carbonic anhydrase 29,000
Trypsin inhibitor 21,000
OxyBlot™ is a trademark of Serologicals Company