The process of western blotting (WB) Gel preparation:
Sample preparation involves complete lysis of your sample of interest. It is important to use fresh samples to generate higher specific signal with lower background levels. The homogenization step should include the proper lysis buffer, certain extractions call for a higher detergent content. Being aware of the problems associated with your starting material, cell line extracts, tissue extracts, or primary cells, will allow for the proper extraction protocol.
Gel electrophoresis using SDS page is the most commonly used technique for separating proteins based on their size and charge. The separation gel and corresponding buffers will also depend on the molecular weights of the target protein(s). To visualize the movement of the protein it is recommended to use a pre-stained marker adjacent to the protein of interest. It is important that there is ample amount of starting sample otherwise there could be a lack of signal due to a low concentration of protein of interest.
Gel transfer from gel to membrane (nitrocellulose, PVDF) can be done by electrophoresis in a buffer solution; this is called a wet transfer method. In contrast, semi dry and dry transfer can also be used. There are variables that can affect the transfer efficiency of each method which can include gel thickness, protein size, and primary antibody selection. If low signal is an issue, it would be recommended to check that total protein lysate per lane was loaded in adequate amount; typically 20-30ug is sufficient, but could be higher based on the starting material e.g. tissue samples.
The next step in the WB sequence is Blot processing. The membrane containing the protein(s) of interest then follows the following sequence that includes: Blocking/washing step, Primary antibody incubation/washing step, Secondary antibody/washing step, detection. The blot processing steps are tedious and time consuming due to the number of steps that involve reagent changes. Since each step in the process is timed, it is virtually impossible to repeat each timed step without automation. Automation leads to reproducible publication quality results. Both the BlotCycler Touch and BlotCycler Mini offer the precise automation necessary for consistent reproducibility, better signal, and lower background.
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