Total Protein Analysis as a Reliable Loading Control for Quantitative Fluorescent Western Blotting
Figure 4
Linear range and sensitivity of total protein stain is greater than the conventional loading controls β-actin or β-tubulin.
(A) Representative LICOR image for a protein dilution series of whole brain homogenate 1, 5, 10, 20, 30, 40 µg demonstrating the working range of β-actin and β-tubulin when using QWB. (B) Quantification of protein dilution series showing the linear ranges of β-actin (black circle) and β-tubulin (open triangle). Note that tubulin expression appears to saturate at less than 10 ug of brain homogenate. (C) In order to pinpoint the saturation level a tubulin specific protein dilution series over a smaller range (0.5, 1, 2, 4, 6, 8, 10,12 and 14 µg) establishing the saturation point of β-tubulin when using QWB. (D) Quantification of β-tubulin linear range. (E) Total protein stain of dilution series 2, 10, 20, 40, 80 µg made using the bovine serum albumin standard (2 µg/µl) from the Pierce BCA kit (see methods). BSA molecular weight is 66.5 kDa. Imaging of this dilution series demonstrates imaging of a broad concentration range without saturation at a single protein mass. (F) Graphical representation of quantification from BSA dilution series in panel E. This demonstrates wide linear detection and high correlation (0.998) validating the use of total protein measurements as a viable method for detecting protein load using the LICOR system. (G) Total protein stain of whole brain homogenate dilution series 1, 5, 10, 20, 30, 40 µg demonstrates the broad concentration range detectable without saturation. (H) Correlation between the total protein stain quantification (red line) and the BCA OD (blue line) for the protein dilution series demonstrates wide linear detection and high correlation (0.996 & 0.979 respectively) validating the use of total protein measurements as a viable “loading control” for QWB using the LICOR system.