OxyBlot Protein Standard

OxyBlot Protein Standard is composed of five proteins (phosphorylase B, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor) containing 1-3 dinitrophenylhydrazone (DNP) residues per protein. These attached DNP moieties permit the visualization of the proteins by the detection system of the OxyBlot Protein Oxidation Detection Kit.

OxyBlot Protein Standard serves as an internal control for the electrophoresis, western transfer & immunodetection steps of the OxyBlot procedure.

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

Store at -25° to -15°C upon receipt. After using for the first time, divide the remaining mixture among several tubes. Dividing the mixture into aliquots serves to protect the mixture from excessive freeze-thaw cycles.

Each vial contains 150 μl of OxyBlot Protein Standard, which is sufficient for 60 minigels.

OxyBlot Protein Standard serves as an internal control for the electrophoresis, western transfer and immunodetection steps of the OxyBlot procedure.

CHEMICON is a registered trademark of Merck KGaA, Darmstadt, Germany

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial