b'SeparateTransferDetectTotal protein normalization with fl uorescent No-Stain Protein Labeling ReagentGet accurate total protein normalizationNo-Stain blot image -ActinThe use of housekeeping proteins for normalization5040302010 5040302010of western blots has its drawbacks, as the expression of housekeeping proteins can vary with experimental conditions, and these proteins can often have oversaturated western blotting signals that interfere withGAPDH5040302010quantitation analysis. Total protein normalization using Invitrogen No-Stain Protein Labeling Reagentavoids the need for housekeeping protein detection, thereby overcoming the variability and inaccuracy of using housekeeping proteins. -Tubulin5040302010FeaturesFlexible-use labeling formatuse with any protein gel to perform total protein labeling of the membrane6Quantitative responseposttransfer, or use it as a fast protein stain afterNo-Stain reagentelectrophoresis of gels you do not intend to transfer 5 - ActinRelative intensity of protein bandGAPDHEasy-to-use and rapid protocolmix and incubate4 - Tubulinwith the transferred PVDF or nitrocellulose membrane or 3gel to label lysine residues; the reaction time is 10 min2Flexible visualizationvisualize with UV or blue LED transilluminators, or by using imaging systems with1fl uorescence (~488 nm) light sources, including iBright ImagingSystems 0 0 10 20 30 40 50 60HeLa cell lysate (g)Accurate total protein normalizationbroad linearTotal protein normalization with No-Stain Protein Labeling range for protein detection of 180 g (total proteinReagent. Bolt 412% Bis-Tris Plus gels were loaded with HeLa lysate load); protein bands are detected down to 20 ng andranging from 10 to 50 g. Proteins from the gels were transferred onto signal is compatible with antibody detection usingPVDF membranes. The PVDF membranes were washed with ultrapure water and labeled with No-Stain labeling solution. The membranes chemiluminescence or fl uorescence methodswere 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.Find out more at thermofi sher.com/no-stain31'