A simple assay for quantification of protein in tissue sections, cell cultures, and cell homogenates, and of protein immobilized on solid surfaces

Abstract

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.

Tumor Necrosis Factor-α Potentiates Intraneuronal Ca2+ Signaling via Regulation of the Inositol 1,4,5-Trisphosphate Receptor

“…All fractionation solutions were spiked with 1ϫ Protease Inhibitor Mixture (Sigma). Fractions were then quantified by an Amido Black assay (15), and 25 g of each sample was then subjected to SDS-PAGE on a 5% gel for the IP 3 Rs or a 12% gel for the surface receptors. Proteins were transferred to polyvinylidene difluoride membranes and incubated overnight with a type-1 IP 3 R antibody (Chemicon, 1:1,000), type-2 IP 3 R antibody, pan-SERCA antibody (AbCam and Sigma, 1:1,000), a pan-PMCA antibody (AbCam 1:1,000), or a TNF-R antibody (AbCam, 1:1,000).…”

5-Azacytidine induces changes in electrophysiological properties of human mesenchymal stem cells

“…Total protein content was determined using the Amido Black method [20]. Protein extracts were run on a 10% SDS-PAGE gel (80 µg total protein from hMSC and 8 µg from P19) and blotted onto a nitrocellulose membrane (BioTrace ® NT, PALL, FL, USA).…”

Advanced Methods for the Investigation of Cell Contact Dynamics in Endothelial Cells Using Florescence-Based Live Cell Imaging

“…For Western blot analysis, the cells were lysed in SDS sample buffer (0.18 M Tris, 6% SDS, 30% glycerin, 80 mg l –1 Bromophenol Blue, 10 mM DTT) followed by determination of the protein concentration in SDS containing sample buffer by the amido black protein assay [15]. SDS samples were separated by 8–10% polyacrylamide gel electrophoresis, wet transferred to a PVDF membrane and exposed to the respective primary antibody followed by incubation with IRDye 600 CW and 800 CW infrared secondary anti-mouse, anti-goat, and anti-rabbit antibodies (1: 10,000, LI-COR Biotechnology, Bad Homburg, Germany).…”