How to optimize antibody concentration for Western blotting?
Each Western blot experiment involves unique antibodies that interact with unique samples under different conditions. There is no single antibody or antigen concentration that is suitable for every experiment. To achieve the most optimal Western blot, antibody concentration needs to be optimized. The ideal antibody concentration depends on experimental conditions such as antigen concentration, antibody specificity and affinity, and buffer composition. Suboptimal antibody concentrations can lead to several common errors including weak signals, nonspecific bands, and speckled or blotchy backgrounds. Optimizing antibody concentration can solve these issues and more, but performing multiple Western blots to find the optimal concentration is time-consuming and wasteful. A simpler, faster, and cheaper method is to switch to a dot blot procedure. 1. Prepare a series of protein sample dilutions with your chosen buffer and prepare antibody dilutions. 2. Cut nitrocellulose membranes into 1 cm long strips, enough to test one or two primary antibody dilutions with two or three secondary antibody dilutions. 3. Spot your samples onto the nitrocellulose strips using as little volume as possible. For samples larger than 5 µl, spot small volumes multiple times at the same point, allowing each spot to dry completely before the next. After spotting, let the membrane strips dry for 10-15 minutes. 4. Block the membranes by soaking in blocking buffer at room temperature for 1-2 hours. Use an orbital shaker to ensure even coverage. 5. Apply the primary antibody dilutions and incubate on an orbital shaker for 1 hour. Strips receiving the same concentration of primary antibody can be incubated together in the same bath. 6. Thoroughly wash the membrane strips in wash buffer. Apply secondary antibody dilutions in the same manner as before. 7. Wash the membrane strips again as in step 6. 8. Prepare the substrate working solution according to the instructions in the datasheet of the substrate you are using. 9. Incubate the nitrocellulose strips with the substrate working solution for 5 minutes or until color develops. 10. When using chemiluminescent substrate, view results or image with film or camera. The optimal protein concentration will produce black dots or a sustained chemiluminescent signal for 5-20 hours.
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