Measuring Protein Concentration on Nitrocellulose and After the Electrophoretic Transfer of Protein to Nitrocellulose

Proteins bind to nitrocellulose membranes when applied directly or after electrophoretic transfer from polyacrylamide electrophoresis gels. Proteins can be stained for visualization with organic dyes Ponceau S, amido black, Coomassie Blue, and colloidal silver/gold and the intensity of the stain is directly proportional to the amount of protein present. Chemicals that interfere with dye/protein interactions in solution can be removed by washing the nitrocellulose after protein application. A method is described whereby protein-dye complexes attached to the nitrocellulose can be solubilized, dissolving the nitrocellulose and releasing dye into solution for detection by a spectrophotometer. The concentration of the dyes Ponceau S, amido black, and colloidal silver is proportional to the concentration of protein. Proteins transferred electrophoretically from SDS-PAGE, isoelectric focusing, or 2D gels to nitrocellulose can be stained with amido black, protein bands excised, and the bound dye detected in a spectrophotometer to quantify proteins in the individual protein bands.