To obtain a reliable Western Blot result , optimizing the concentration of antigen and antibody to be used in the assay is a fundamental step.
There is no specific optimal concentration that can be applied to all experiments, since the rate of antigen-antibody binding will be conditioned by factors such as the amount of antigen present in the sample, the specificity of the antibodies, the temperature, the pH or the buffers components.
In this post, we will review a simple way to optimize the concentration of antigen and antibody for western blotting , and thus avoid problems such as weak or non-existent signals, nonspecific bands or excessive background noise, among others.
OPTIMIZE ANTIGEN AND ANTIBODY CONCENTRATION FOR WESTERN BLOT
The optimal antigen and antibody concentration can be determined by running several Western Blots at different concentrations. But there is a much easier and faster way, and that is by performing a dot blot.
What would be the procedure to optimize the antigen and antibody concentration using a dot blot? Below we summarize the steps to follow:
1.- Prepare dilutions from the protein sample in PBS or TBS.
2.- Cut a strip of nitrocellulose membrane (approximately 1cm) for each concentration of primary or secondary antibody that you want to test.
3.- Place the dry nitrocellulose membrane strips on filter paper and place spots of each antigen dilution on the strips, always using the smallest possible volume in each one.
4.- Let the membranes dry.
5.- Block the non-specific sites of the nitrocellulose membranes by incubating them with the blocking buffer at room temperature and with agitation.
6.- Prepare dilutions of the primary antibody and apply it to the membrane strips.
7.- Incubate again at room temperature with shaking.
8.- Wash the membrane strips with the washing buffer.
9.- Prepare dilutions of the secondary antibody and add them to the membrane strips.
10.- Re-incubate at room temperature with shaking.
11.- Wash the membranes again with the washing buffer.
12.- Prepare the solution with the substrate .
13.- Incubate the membrane strips with the substrate solution.
14.- Remove the membrane strips and place them on plastic sheets or some other protective wrapping .
15.- With the protein part facing up, place the wrapped strips against the film and expose them for 30-60 seconds .
16.- An optimal blot should result in a clear band for the protein of interest with no background signal or unspecific bands.
17.- In the event that the optimal results were not achieved, the entire process should be repeated, testing different dilutions of antigen and antibody.