![]() 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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