Essential problems in quantification of proteins following colloidal staining with Coomassie Brilliant Blue dyes in polyacrylamide gels, and their solution

Abstract

Quantitative determination of stained proteins following polyacrylamide gel electrophoresis (PAGE) is of increasing interest especially since computer-aided densitometers have become available as well as recipes for sensitive and background-free staining with Coomassie Brilliant Blue dyes. However, avoidance of separation artifacts is not the only essential prerequisite for quantitative evaluation. The local particle density of a protein in a given gel is of critical importance since it determines its stainability. Depending on local protein concentration, the dye binding to the same amount of a given protein differs considerably. Since the stainability of proteins using colloidal staining procedures, as with Coomassie Brilliant Blue dyes, is time-dependent and, in addition, also dependent on the pore size of a given polyacrylamide gel used for PAGE, calibration curves for quantitative determinations have to be prepared in polyacrylamide gels of the same composition as used for PAGE. Staining conditions also have to be identical for calibration gels and gels under analysis. If, however, a set of calibration curves is prepared for different staining times, it is possible to calculate a generalized calibration curve, allowing for quantitative evaluation with flexible staining time. Furthermore, and in consequence of the implications due to particle density, quantitative determination via densitometry is only possible by determining the protein amount of each single measuring point (pixel) via its absorbance on the basis of a calibration curve. Since the particle density is inherent in a calibration curve, the final summation of the protein amount per pixel will give values close to reality.

Differential antiproliferative mechanisms of novel derivative of benzimidazo[1,2-α]quinoline in colon cancer cells depending on their p53 status

“…Studied quinoline derivative has stronger cytostatic activity than the antimetabolite 5-fluorouracil (IC 50 , 10 F 1.4 and 6.7 F 1.1 Amol/L in HCT 116 and SW620, respectively) 6 and quite similar to a topoisomerase I inhibitor irinotecan [IC 50 , 2.0 F 0.9 Amol/L in SW620 (46) and 2.2 Amol/L in HCT 116 (47)], standard agents in colorectal cancer therapy. These comparison data shed new light on the role of quinoline derivative as promising, novel agent against colorectal cancer.…”

“…Obtained gels were stained with Colloidal Coomassie Blue (Bio-Rad; ref. 6) and scanned by the VersaDoc Imaging System 4000 (Bio-Rad). Image analysis was carried out using PDQuest software version 7.0 (Bio-Rad), whereby total density in gel image was used as normalization method.…”

The Role of Hydrogen Peroxide-Producing and Hydrogen Peroxide-Consuming Peroxidases in the Leaf Apoplast of Cowpea in Manganese Tolerance

“…The 2-D gels were stained with colloidal Coomassie Blue (Neuhoff et al, 1985(Neuhoff et al, , 1990. Gels were fixed in 40% (v/v) MeOH/10% (v/v) HAc for at least …”

High‐throughput negative detection of SDS‐PAGE separated proteins and its application for proteomics

“…Currently, the most common methods for universal profiling of proteins in gels are Coomassie brilliant blue, silver and fluorescent stainings. Although Coomassie brilliant blue stain is a reproducible, low-cost, and MS compatibility method, but it is relatively insensitive and time-consuming [2][3][4]. Silver staining is the most sensitive staining method for the visualization of protein in gels, up to 100 times more sensitive than Coomassie brilliant blue staining, but it lacks reproducibility and shows poor linearity with protein concentration [5][6][7][8][9].…”