Western Blotting is a universal technique that is used to target the presence of peptides and proteins in complex biological mixtures. SDS page is used to separate proteins based on size, followed by a transferring to a support membrane, and visualized by using a primary & secondary antibody. The question is how do you perform this technique with minimal user error and repeatable reproducibility. Although, the technique is widely used problem can occur that lead to suboptimal results. Here are a few suggestions and Quick Tips that should help:
- Make sure all your lab equipment is clean. Any contamination can cause poor results. No one like to waste reagents and a days worth of work to end up with poor results.
- Be careful of your loading volume and concentration. Over loading can cause a host of problems: lane bleeding, migration distortion, background issues and the blot can be difficult to read.
- If you are using HRP conjugated antibodies do not use azide unless you want to inactivate the enzyme
- You should validate your primary and secondary antibodies. If they don’t detect the target under your experimental conditions you have a problem.
- How do you know the correct concentration of antibodies to use in your experiment? You don’t unless time is taken to titrate your antibody. An initial experiment can be run at various antibody concentrations to determine the best concentration. Commercial antibodies can vary from lot to lot therefore titration is an important step. The problem with antibody optimization is the time necessary to determine the ideal concentration. Precision Biosystems has developed a one-step protocol to perform a serial dilution of six different antibody concentrations in a fully automated fashion using the BlotCycler Touch. We also recommend preparing your antibody the day of your experiment.
- Be careful selecting your blocking solution. For example, using a normal serum from the host species from which your secondary antibody was derived can eliminate background. Also blocking the membrane for too long can cause a reduced binding interaction with the protein of interest.
- The proper use of stripping buffer can be overlooked. Precision Biosystems stripping buffer( cat# SB200) has been optimized for efficient and fast removal of primary and secondary antibodies leaving the antigen attached to the membrane and ready for the next immunodetction.
- When working with tissues or cell extracts stick with an antibody that has minimal cross reactivity . If not, high background can be a problem.
- Do all washing steps at 4 degrees. Based on the literature and our customers observations washing at 4 degrees produces higher signal and less background and helps eliminate non-specific bands.
- Automation is recommended for all your blot processing steps. The hands free operation of the BlotCycler Touch and Mini eliminates user error, optimizes blocking, shaking, washing, and antibody incubation. Both instruments produce high quality reproducible results.
