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

  • J. P. Dean GoldringEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1314)


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.

Key words

Protein staining Amido black Ponceau S Silver Nitrocellulose Protein concentration 


  1. 1.
    Goldring JPD (2015) Spectrophotometric methods to determine protein concentration. In: Kurien BT, Scofield RH (eds) Western blotting, Methods in molecular biology. Springer, New YorkGoogle Scholar
  2. 2.
    Noble JE, Bailey JA (2009) Quantitation of protein. In: Burgess RR, Deutscher MP (eds) Guide to protein purification, vol 463, Methods in enzymology. Academic, New YorkGoogle Scholar
  3. 3.
    Goldring JPD, Ravaioli L (1996) Solubilization of protein-dye complexes on nitrocellulose to quantify proteins spectrophotometrically. Anal Biochem 242:197–201PubMedCrossRefGoogle Scholar
  4. 4.
    Neumann W, Habermann E, Amend G (1952) Zur papierelektrophoretischen fraktionierung tierischer gifte. Naturwissenschaften 39:286–287CrossRefGoogle Scholar
  5. 5.
    Schaffner W, Weissmann C (1973) A rapid, sensitive and specific method for the determination of protein in dilute solution. Anal Biochem 56:502–514PubMedCrossRefGoogle Scholar
  6. 6.
    Heda GD, Kunwar U, Heda RP (2014) A modified protein assay from microgram to nanogram levels in dilute samples. Anal Biochem 15:67–72CrossRefGoogle Scholar
  7. 7.
    Choveaux D, Krause RDE, Goldring JPD (2012) Rapid detection of proteins in polyacrylamide electrophoresis gels by Amido Black and Direct Red 81. In: Kurien BT, Scofield RH (eds) Protein electrophoresis: methods and protocols, vol 53. Springer, New York, pp 585–589CrossRefGoogle Scholar
  8. 8.
    Aldridge GM, Podrebarac DM, Greenough WT, Weiler IJ (2008) The use of total protein stains as loading controls: an alternative to high-abundance single-protein controls in semi-quantitative immunoblotting. J Neurosci Methods 172:250–254PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Meola JM, Vargas MA, Brown HH (1997) Simple procedure for measuring total protein in urine. Clin Chem 23:975–977Google Scholar
  10. 10.
    Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Golda JE, Gomes AV (2013) Stain-free total protein staining is a superior loading control to β-actin for Western blots. Anal Biochem 440:186–188CrossRefGoogle Scholar
  12. 12.
    Solomon JV (1851) Observations of nitrate of silver stains of the conjunctiva. Case of absolute blackness. Prov Med Surg J 15:594PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.BiochemistryUniversity of KwaZulu-NatalScottsvilleSouth Africa

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