We’ve all been there before…. You worked hard all day and think that you have executed a brilliant experiment, only to realize that you generated data that looks like a hot mess. This is the worst feeling ever! The worst part is that there is literally nothing that you can do about it now. There are just not enough hours in the day to recover from your epic flop. All you can do now is go home and think about how you are going to explain to your PI that you accomplished nothing yesterday. Before this happens again, take a minute to carefully think about five critical pieces of the Western Blot puzzle.
I. Protein Samples
Unfortunately, not all proteins are created equally. Take the time to really get to know your protein. Knowing its cellular location and general properties will help you quickly optimize the protein extraction buffer. Once you have extracted your protein, don’t forget to properly quantitate it using a protein assay such as the Bradford or BCA assay. Loading the same amount of protein in each well is critical for Western Blot Success.
Whether you like to cast your own gels or buy commercial ones, always check for signs that your gel is not up to par:
- Collapsing lanes
- Noticeable bubbles
- Consistency-the gel should not be dry or runny along the edges
- Double check the expiration date and percentage of commercial gels
- Protein ladder does not migrate as expected during electrophoresis. Cut your losses here, if the ladder is not migrating correctly there is a high probability that your target isn’t either.
- Protein ladder does not transfer evenly from the gel to membrane. If you can’t see all the bands of your ladder you may not be able to see your target either. This is a good time to Ponceau stain your membrane to confirm.
Antibodies can often be troublesome if the affinity and specificity for the target are not high. Most of the time this will be out of your control. Here are things you can control:
- Properly screen and validate your antibody for specificity
- Titrate the primary and secondary antibodies. Using too much antibody is a major source of nonspecific banding and high general background.
- Use pre-adsorbed secondary antibodies for the highest specificity
- Choose a blocking buffer that does not interfere with your antibodies
The use of a high quality substrate is extremely important for day-to-day reproducibility. Using inconsistent substrates will leave you frustrated at the end of your day. Many suppliers offer substrates with different sensitivities. Start by choosing the substrate that is appropriate for the abundance of your protein. Using a high sensitivity substrate for a protein that is highly abundant does not make sense and will only cause trouble as it will result in saturated bands.
Try some of Advansta’s high quality reagents to ensure that your next Western story ends happily ever after…
AdvanBlock™-Chemi blocking solution:
HRP Conjugated Secondary Antibodies:
AdvanWash washing solution: