Antibody selection for Western Blotting is a critical step. To avoid wasting valuable samples, time and funds, selection of the correct antibody for Western Blotting is critical. A good start is to review the data sheet provided by the manufacturer including whether the antibody has been validated. Journals tend not to include antibodies with insufficient information and validation effort. Making sure that the antibody is validated for a specific species is also important. The primary antibody clonality is also an additional characteristic to consider. Polyclonal and Monoclonal antibodies have advantages and disadvantages. Depending on your target protein either could be suitable. Monoclonal antibodies have high specificity and low cross reactivity due to a single epitope; the downside is they are less likely to work across different species. If epitope changes occur in the target protein less binding could occur. Another consideration is that Monclonal antibodies are typically more expensive that Polyclonal antibodies. Depending on your experimental needs a Polyclonal antibody could suffice; Polyclonal antibodies posses multiple epitope protein recognition sites, which results in a higher binding affinity to target proteins. The downside could be cross-reactivity due to multiple epitopes. Your primary antibody incubation should be done a 4 degrees overnight for best results. Automating the blot processing steps using the Precision Biosystems BlotCycler improves results and provides the following benefits:
Eliminate variations in fluid delivery and timing
Get reproducible results, repeatedly
Perform membrane processing precisely, every time
Program your protocol, and walk away
Process up to 24 membranes simultaneously
Get precise publication-quality results time after time.
https://precisionbiosystems.com/
