Western Blot Antibody Concentration Optimization

How to optimize antibody concentration for western blotting?

Each Western blot experiment involves a unique antibody interacting with a unique sample under varying conditions. No single antibody or antigen concentration will work for every experiment. In order to obtain the most perfect Western blots, the antibody concentration needs to be optimized. The ideal antibody concentration is dependent on the concentration of antigen, the specificity and affinity of the antibody, and experimental conditions such as buffer composition.

Suboptimal antibody concentration can cause several common errors, including weak signal, nonspecific bands, and flecked or blotched background. Optimizing antibody concentration can resolve these problems and more, but performing multiple Western blots to obtain the optimal concentration is time consuming and wasteful. An easier, quicker, and cheaper method is to use a dot blot assay instead.

Dot Blot Protocol

  1. Prepare a range of dilutions of your protein sample using the buffer of your choice, and prepare your antibody dilutions.
  2. Cut a nitrocellulose membrane into 1cm strips, enough to test one or two primary antibody dilutions with two or three secondary antibody dilutions.
  3. Dot your samples onto the nitrocellulose membrane strips using as little volume as possible. To dot samples greater than 5ul, dot smaller volumes multiple times on the same spot, allowing each dot to dry completely each time. After dotting is done, allow the membrane strips to dry for 10-15 minutes.
  4. Block the membrane by soaking in blocking buffer for 1-2 hours at room temperature. Use a shaker to ensure even coverage.
  5. Apply the primary antibody dilutions, incubating for 1 hour on an orbital shaker. The strips that are receiving the same concentration of primary antibody may be incubated together in the same bath.
  6. Wash the membrane strips thoroughly in wash buffer.
  7. Apply the secondary antibody dilutions in the same manner as before.
  8. Wash the membrane strips again, as in step 6
  9. Prepare the substrate working solution as described in the data sheet for the substrate you are using.
  10. Incubate the nitrocellulose strips with the substrate working solution for 5 minutes, or until color appears.
  11. Observe the results, or image with film or camera when using chemiluminescent substrate. Optimal protein concentrations will result in dark dots or strong chemiluminescent signal that lasts 5-20 hrs.

Keywords: Western blotting optimization, antibody concentration, dot blot assay, best antibody concentration

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