Western Blotting is a common, but lengthy procedure composed of many small, simple steps that add up to a complex process. Mistakes in a major or several of the small steps can lead to a poor quality image or no data at all. As with any complex process, the key to success lies in planning and focusing on the tasks at hand. Below are some considerations to help in building your Western Blot experimental plan.
Optimize: Have you done your homework?
The overall procedure for Western Blotting, depending on how you want to slice it, may be broken down into several ‘sub-routines’: protein extraction/sample preparation, electrophoresis, transfer, blocking & antibody incubation; imaging; and analysis. There are numerous generalized protocols that will provide you with reasonable confidence in success. However, to be cliche, every protein is different and may require specific conditions to achieve that masterpiece image. Is milk or BSA-based blocking solution best? What size is your protein? Prior to starting, thoroughly read the work of others who may be working with your protein or a protein similar in function, structure, & size. Use their protocols as starting guides. It is guaranteed you will have that one protein that requires its own unique set of conditions. So, get optimizing.
Antibodies: Are you sure it is the correct one?
Avoid some common mistakes such as making sure both the primary and secondary Ab are for the correct species. For primary Abs, some may be more specific for human or mouse, e.g. Read the fine print of the data sheets. If you are working with a rabbit-derived primary antibody, be sure that your secondary antibody is anti-rabbit. You will likely find several Ab sources for your protein. Test several primary Abs to see which one provides the greatest specificity and sensitivity.
Incubations: A little shake with that roll (or vice versa).
Optimized shaking conditions during incubations can speed up the overall process, reduce background, and increase sensitivity. Ideally, if you only roll or shake, you want to add the other motion to ensure the Abs thoroughly diffuse and interact with the membranes.
Imaging: Don’t be satisfied with just one.
When you are starting to perform westerns or working with a new Ab, bracket your exposures for different time periods so that you may determine the optimal exposure time. Start with the longest time period first and work-down.
Membranes: Nitrocellulose or PVDF?
Largely, this decision comes down to personal preference. Generally, nitrocellulose is less expensive and many believe it provides a higher sensitivity. Potential down sides are that NC is more fragile; and, un-supported NC is not amenable to re-use. PVDF does not tear or break easily, can be stored and re-probed. It does require a methanol 'charge'.
Be clean! If you aren't, who will?
As with any common equipment or bench areas, western equipment and bench space can become pretty gross. Everyone thinks the next person should be the one to clean, so they just give it a cursory rinse and head to journal club. It is not fair that you are always the one who seems to get stuck with the thankless task. However, you have a choice – good results or not? Before and after, thoroughly clean your apparatus pieces, remove any stains, clean up dried spill residue in common areas, etc. Leave it better than you found it.
At VitaScientific, we offer several products from unique mixers to standard blue-film that will help you achieve that publication quality western. How may we help you?
