Purity estimation experiment by overloaded stained SDS gel scanning method

Summary

Since proteins can be separated with high resolution on SDS gels and sensitive staining can be given with Caumas Brilliant Blue (CBB), the purity of the protein samples can be obtained by scanning the protein preparations separated by SDS gels after staining and destaining as described in Experiment 4 of this unit. This experiment is from the Experimental Guide for Protein Purification and Identification, by Zhu Houzhu.

Operation method

Purity estimation experiment by overloaded stained SDS gel scanning method

Materials and Instruments

CBB Staining Solution Decolorizing Solution

Move

reagents

CBB Stain

Decolorizing solution

(For the recipe, see "Preparation of Reagents", PP.184~189)

Procedure

1) As described in Experiment 4 (pp.159~160), take several diluted samples of pure protein (containing about 1, 3, 10 and 30ug of total protein), and electrophoresis, staining and destaining on small SDS-polyacrylamide (10%) gels.

2) The decolorized gel is scanned and the amount of dye conjugate contained in the primary band (presumably the target protein) and each of the secondary bands (presumably the obvious heteroproteins) is determined.

3) Assuming that all proteins bind the same amount of CBB per microgram, calculate the relative amounts of the primary and secondary bands accordingly. Some very small amounts of heteroproteins are visible only on overloaded gel lanes, and not when the total protein spiked is only 1ug.

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