A Ponceau Staining based Dot-Blot Assay for reliable and cost-effective Protein Quantification

Abstract

Reliable quantification of protein extracts from tissues can be a challenge e.g. due to interference of the high fat content in tissues of the nervous system. Further problems like under- or overerstimation of protein concentrations in protein quantification kits like the bicinchoninic acid (BCA) assay can occur. In addition, common lysis buffers such as RIPA buffer are known to be unable to solubilize a large amount of proteins (~10-30%) leading to unsatisfactory and unreliable experimental results with techniques such as immunoblotting. In this work, we have developed a Ponceau S staining based protein quantification assay. This assay is compatible with tissues or cells directly lysed in 2x SDS gel loading buffer, containing bromophenolblue, leading to more complete protein extraction. Protein concentrations of several samples can be determined in a fast and cost-effective manner and subsequent experiments (e.g. Western blot) can be performed without loss of proteins. The presented protein quantification method is highly reliable, fast and economical. Using this method, it is possible to save between 2300 to 3200{euro} per 1000 lysates as compared to the costs of a commercial BCA kit.