A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces.
The determination of total protein is often a key step for the quantitative analysis of various parameters in tissue and general biochemical research. The classical protocols are restricted to a few compatible buffers, and protocols for the determination of protein in solutions containing protein agglomerates or of protein immobilized on solid surfaces are not available. In such cases, quantification may be complicated. Here, we describe a simple sensitive method for protein quantification circumventing all these restrictions. Proteins in solution or suspension in any buffer are spotted onto cellulose acetate, dried, and stained with Amido Black. After washing off the excess dye, bound Amido Black is solubilized in an acidic solution and determined photometrically. Tissue slices (fixed or native), adherent cell cultures, or Western blots can also be stained and their protein content determined irrespective of the supporting material. A micro-version of the protocol for proteins in solution allows large numbers of samples to be evaluated at a time in microtitration plates and requires only 1-2 microl per sample. A linear concentration dependency (r2=0.950-0.999) was obtained for all samples in all cases investigated. The method presented here permits the exact determination of soluble protein in a large variety of buffers, of insoluble or immobilized protein present on a wide variety of supports, and even of whole cells or tissue slices.
Dieckmann-Schuppert
A
A
Zentrum für Hygiene und Medizinische Mikrobiologie, Arbeitsgruppe Parasitologie, Philipps-Universität Marburg, Robert-Koch-Strasse 17, D-35037 Marburg, Germany.
Schnittler
H J
HJ
D000580:Amido Black / Q000737:chemistry*; D000818:Animals; D002021:Buffers; D002478:Cells, Cultured; D004396:Coloring Agents / Q000737:chemistry*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000378:metabolism; D008867:Microtomy; D011506:Proteins / Q000032:analysis*; D051381:Rats; D013057:Spectrum Analysis; D013194:Staining and Labeling / Q000379:methods*; D014471:Umbilical Veins / Q000166:cytology / Q000378:metabolism
D002021:Buffers; D004396:Coloring Agents; D011506:Proteins; D000580:Amido Black