The blocking agent was not well dissolved. Keep shaking when incubating the antibody. Reduce the voltage to slow down the migrationĭecrease the secondary antibody concentrationĪir bubbles were trapped against the membrane during transferring or the antibody is not well distributed during incubation Migration was too fast during electrophoresis Substrate is not well-distributed during incubation Remove bubbles in the gap of gel and membrane when preparing for transferring. Modification of proteins, such as glycosylation or phosphorylation, can result in an increase of molecular weight of protein.Īir bubbles were trapped in the gap of gel and membrane during transferring. Increase a process or intensity of protein denaturation Run a secondary antibody control or choose other secondary antibody Non-specific signal caused by the secondary antibody The size of every isoform protein is different. Some proteins derived from the same gene have different isoforms. Use primary cells or less passaging cells to run a controlĭecrease the concentration of the primary or secondary antibodyĬhoose monoclonal antibody or affinity purified antibody to ensure antibody specificityĮxistence of the different protein isoforms Use fresh samples and use protein inhibitorĬells were cultured too many passages to result in protein variation Setting loading control can validate the secondary detecting system.Īvoid sodium azide in all solutions and containersĭirectly mix enzyme and substrate. Primary antibody and species, primary antibody and secondary antibody, or enzyme and substrate are not compatible. The reagents are not compatible with each other

Increase the concentration of the antibody and the incubation time. Insufficient reaction of antibody to membrane Use effective antibody in expiration, avoid freezing- thawing repeatedly, and use fresh solution. Lower the concentration of your blocking solution and shorten blocking time. As a result, choose suitable methanol concentration according to different molecular weight. At the same time, it may cause shrinking or hardening of the gel to inhibit transferring of high molecular weight proteins. Too high concentration of methanol may result in the separation of protein and SDS and thus cause protein precipitation in the gel. Control transfer temperature and optimize transfer electricity and time. Always ensure assembling electrode correctly. Make sure there are no air bubbles between the gel and membrane during transfer. Use 0.2 µm size membrane for proteins smaller than 22 KD. Use 0.45um size membrane for proteins larger than 22KD. If the level of target protein in samples is low, try to increase amount of loading sample.Ĭhoose suitable pore size membrane. No or low level of target protein in samples Optimize blocking solution, decrease blocking time or decrease the concentration of proteins in the blocking solution. The antigen is blocked by blocking buffer Add Tween-20 to the washing buffer to reduce cross reaction. The antibody concentration may be too highĭecrease the concentration of primary antibody or secondary antibodyĬross-reaction between antibody and blocking agentĬhoose blocking solution without cross-reaction.

Increase washing time and washing buffer’s volume Incubate in sufficient reaction solution to prevent the membrane from drying out. Membrane-compatible – colored bands transfer to membranes for Western blottingĭye-stained proteins in 62.5mM Tris-H 3PO 4 (pH 7.5 at 25☌), 1mM EDTA, 2% SDS, 10mM DTT, 1mM NaN 3 and 33% glycerol.Use clean tweezer and operate with gloves to prevent membrane fouling.Quality tested – each lot evaluated by SDS-PAGE and Western blotting.Sharp bands – color-coded bands of similar intensity for easy visualization.Ready-to-use – supplied in a loading buffer for direct loading on gels no need to boil.Multicolor – four different colors for unambiguous band-size assignment.Size range – 10 proteins spanning 10 to 260kDa Four different chromophores (blue, orange, green, pink) are bound to the different component proteins, producing a brightly colored ladder with an easy-to-remember pattern. This prestained protein MW marker is designed for monitoring the progress of SDS-polyacrylamide gel electrophoresis, for assessing transfer efficiency onto PVDF, nylon and nitrocellulose membranes, and for estimating the approximate size of separated proteins that have been made visible with gel stains or Western blot detection reagents. Thermo Scientific™ Spectra Multicolor Broad Range Protein Ladder is a 4-color protein standard containing 10 prestained proteins (10 to 260kDa) for use as size standards in gel electrophoresis and Western blotting.

Western blot no protein or ladder