![]() ![]() (A) After blocking, the membrane was analyzed by western blot using SuperSignal West Dura Extended Duration Substrate. Three different concentrations of HeLa cell lysate were separated by SDS-PAGE and transferred to a to 0.45 μm nitrocellulose membrane. ![]() Comparison of stripping buffers on nitrocellulose. The bond between streptavidin and biotin is very strong and can be difficult to dissociate for reprobing. Minimize these effects by probing for low-abundance proteins or with low-affinity antibodies first. With every round of stripping, there is a potential for antigen loss. Use more stringent conditions if required to break up strong antigen-antibody pairs. Start with gentle, mild conditions to avoid loss of sample. Strip and reprobe only chemiluminescent and fluorescent western blotsĬolorimetric/chromogenic detection reagents leave a permanent visible stain on the membrane that will not be removed by stripping procedures. PVDF is less brittle and fragile than nitrocellulose, making it more useful when requiring multiple rounds of reprocessing (stripping and reprobing procedures). PVDF membranes have a protein binding capacity of 170–200 µg/cm 2 and offer better retention of adsorbed proteins than other supports because of the greater hydrophobicity. By stripping the membrane, the blot can be reused. Immunoblotting requires many steps, providing ample opportunity for mistakes to occur. When immunoblot results are not as expected, reprobing allows the use of the same protein sample without going back to gel electrophoresis. Optimization is achieved easily by stripping the membrane and reprobing with a different antibody concentration. The light emission intensity of high sensitivity chemiluminescent substrates often require antibody concentration optimization to achieve the highest quality blot. By using the same blot for several different detections, you save time.īy reusing the same blot, you save money on the costs of the gel, membrane, and protein sample. It is time-consuming to run an SDS-polyacrylamide gel and then transfer the proteins to a membrane. When the protein mixture is rare or valuable, reprobing conserves the sample and allows the membrane to be analyzed with the same or different antibodies. ![]()
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