A Quick and Easy Method to Separate Proteins of Similar Molecular Weights Using 2D Electrophoresis

Two-dimensional electrophoresis is a critical technique for proteomic research; many researchers believe two-dimensional (2D) electrophoresis can only be accomplished using a combination of 2D gel electrophoresis

to separate proteins, followed by visualisation through either (a) mass spectrometry (MS) or (b) the use of a cooled CCD imaging system combined with image analysis software for protein identification. Although these

techniques are powerful and sensitive, proteins can be identified easily and quickly by staining a 2D gel with either Coomassie blue or silver stain. 2D gels were used to determine a quick method to differentiate human

alpha-NAGAL protein from IGG antibodies (with similar molecular weights) contaminates obtained during fractionation.

MATERIALS AND METHODS

IPG BlueStrips were from Serva Electrophoresis GmbH (Heidelberg, Germany). CodeBlue stain was from Pierce (Rockford, IL). All other reagents used were from Hoefer, Inc (Holliston, MA).

Preparation

Samples of Human alpha-NAGAL protein, IGG antibodies, and a mixture of both were loaded onto 7cm (3-10NL) immobilized pH gradient (IPG) strips using overnight rehydration. Three samples were prepared; (a)10μg of pure alpha-NAGAL protein (b)10μg of rabbit IGG (c)5μg of pure alpha-NAGAL protein and 5μg of rabbit IGG. Each sample was mixed in 130μls of fresh rehydration buffer (Table 1) and then 130μl of rehydration buffer was added to each channel of the rehydration tray (Table 2).

Each sample was placed on different IPG strips, the IPG strips were placed gel side down in the rehydration buffer and then overlaid with mineral oil to prevent evaporation during the overnight rehydration at room temperature.

Note - It is important not to place the IPG strips into a refrigerator or the urea will crystallise.