Quantitation of electrophoretic eluted proteins.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Classical Lowry and Bradford protein assays face interferences when quantifying stained, electroeluted proteins.
• Substances interfering with the Lowry method are resistant to trichloroacetic acid precipitation.
• Bradford assay interferences include detergents, pH, Coomassie brilliant blue R250, and potentially acrylamide fines.

Purpose of the Study:

• To identify and overcome interferences in Lowry and Bradford protein assays for electroeluted samples.
• To establish reliable methods for accurate protein quantitation after electroelution.
• To enable the use of Bradford and Lowry assays within specific microgram ranges for processed samples.

Main Methods:

• Investigated interferences in Lowry and Bradford assays with electroeluted proteins.
• Evaluated trichloroacetic acid precipitation for removing Lowry interferences.
• Assessed compensation strategies for Bradford assay interferences (blanks, standard curves).
• Implemented concentration and dialysis for sample preparation prior to protein quantitation.

Main Results:

• Lowry assay interferences could not be removed by trichloroacetic acid precipitation.
• Bradford assay interferences from detergents, pH, and Coomassie blue could be compensated, but acrylamide fines remained problematic.
• Concentration and dialysis successfully removed interfering substances from electroeluted samples.
• Accurate protein quantitation was achieved using Bradford assay in the 1-20 microgram range and Lowry assay in the 1-20 microgram range.

Conclusions:

• Concentration and dialysis are crucial steps to overcome interferences in protein quantitation of electroeluted samples.
• These methods enable the reliable application of Bradford and Lowry protein assays for specific ranges of electroeluted proteins.
• Successful applications demonstrated for bovine serum albumin, human hexokinase, and phosphoglucomutase.