b'No-Stain Protein Labeling ReagentThe use of housekeeping proteins for normalization of western blots has its drawbacks, as the expressionNo-Stain blot image -actinof housekeeping proteins can vary with experimental5040302010 5040302010conditions, and these proteins can often have oversaturated western blotting signals that interfere with quantitative analysis. Total protein normalization using Invitrogen No-Stain Protein Labeling Reagent avoidsGAPDH5040302010the need for housekeeping protein detection, thereby overcoming the variability and inaccuracy of using housekeeping proteins for normalization.Features: -tubulin5040302010 Flexible-use labeling formatuse with any protein gel to perform total protein labeling of the membrane post-transfer, or use it as a fast protein stain after electrophoresis of gels you do not intend to transfer 6Quantitative response Easy-to-use and rapid protocolmix and incubate5 No-Stain reagentwith the post-transfer membrane (PVDF or nitrocellulose)- ActinRelative intensity of protein bandGAPDHor gel to label lysine residues; the reaction time4 - Tubulinis only 10 min3 Flexible visualizationvisualize with UV or blue LED transilluminators, or by using imaging systems with2fluorescence (~488 nm) light sources, including iBright ImagingSystems 1 Accurate total protein normalizationbroad linear0 0 10 20 30 40 50 60range for protein detection of 180 g (total protein load;HeLa cell lysate (g)Figure 47); protein bands are detected down to 20 ng Figure 47. Total protein normalization with No-Stain Protein Labeling and signal is compatible with antibody detection usingReagent. Bolt 412% Bis-Tris Plus gels were loaded with HeLa cell lysate chemiluminescence or fluorescence methods ranging from 10 to 50 g. Proteins from the gels were transferred onto PVDF membranes. The PVDF membranes were washed with ultrapure water and labeled with No-Stain labeling solution. The membranes were then washed with ultrapure water, followed by immunoblotting for -actin, GAPDH, and -tubulin antibodies, followed by goat anti-mouse Alexa Fluor Plus 680 antibody. The blot was imaged and analyzed with the iBright FL1500 Imaging System. The linear regression value for the entire concentration range using the No-Stain reagent was R = 0.9990, whereas the R values for -actin, GAPDH, and -tubulin were 0.8851, 0.9438, and 0.8332, respectively.Learn more at thermofisher.com/no-stain69'