title: A modified protein assay from microgram to low nanogram levels in dilute samples.
abstract: In this article, we present a modified and improved protein assay that was previously described as "amidoschwarz assay" by Schaffner and Weissmann. Our improved protein assay is user-friendly and 30-40 times more sensitive than the earlier method. The assay was developed into three formats (macro-, micro-, and nanoassay) with trichloroacetic acid (TCA) as protein precipitating agent, measuring up to 96 samples. The macro and micro formats of this assay require a single reagent staining with amido black of protein dots bound to nitrocellulose membrane with lowest protein measurements to 1 and 0.1 μg, respectively. On the other hand, the nanoassay, with combination staining of amido black followed by colloidal gold, can extend the detection limit to 2.5 ng of protein. Protein concentrations were determined by densitometry and/or spectrophotometry. This assay is compatible with many ionic and non-ionic detergents. This improved protein assay provides an additional choice to researchers in measuring total protein concentration accurately in dilute biological samples as low as 0.125 μg/ml prior to their biochemical analysis such as in comparative proteomics.
authors.affiliation: Department of Sciences and Mathematics, Mississippi University for Women, Columbus, MS 39701, USA; Department of Medicine, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA. Electronic address: gheda@as.muw.edu.
authors.forename: Ghanshyam D
authors.initials: GD
authors.lastname: Heda
authors.forename: Upasana
authors.initials: U
authors.lastname: Kunwar
authors.forename: Rajiv P
authors.initials: RP
authors.lastname: Heda
mesh_terms: D000580:Amido Black / Q000737:chemistry; D000818:Animals; D002417:Cattle; D003101:Collodion / Q000737:chemistry; D003720:Densitometry; D003902:Detergents / Q000737:chemistry; D017979:Gold Colloid / Q000737:chemistry; D008567:Membranes, Artificial; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D013053:Spectrophotometry; D014238:Trichloroacetic Acid / Q000737:chemistry
chemical_list: D003902:Detergents; D017979:Gold Colloid; D008567:Membranes, Artificial; D011506:Proteins; D012710:Serum Albumin, Bovine; D014238:Trichloroacetic Acid; D003101:Collodion; D000580:Amido Black
keywords: Amido black; Colloidal gold; Densitometry; Nitrocellulose membrane; Protein assay; TCA