Although Ponceau cannot be used to identify a specific protein of interest, the presence of many faint pink/red bands on the blotting membrane confirms that proteins have been separated through the gel and have transferred onto the membrane. To confirm the transfer of proteins from the gel onto the blotting membrane, Ponceau S reversible stain can be a used before the blocking step. If not, then the transfer of the proteins from the electrophoresis gel to blotting membrane may have been unsuccessful. The prestained protein marker or ladder should be visible on the membrane after transfer. No bands are visible on the blotting membraneĬan the protein marker be seen on the membrane? If the Western blot is not behaving as expected, our troubleshooting guide may help isolate the problem. The robust nature of the antigen-antibody interaction allows the presence of specific proteins and peptides to be detected from complex mixtures. There are aggregates in the HRP conjugated secondary antibody.įilter the secondary antibody agent and remove the aggregates.Download PDF Western blotting is a staple technique of the molecular biology lab. The antibody reacts with the blocking solution. 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. Use clean tweezer and operate with gloves to prevent membrane fouling.
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