Western blot troubleshooting
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Western blot troubleshooting blank film – primary antibody problems
This western blot protocol assumes you have read and completed Step One for troubleshooting blank film. If you haven’t don’t write us and tell us this protocol doesn’t work. If you have, you no longer have “blank film”.
If you don’t follow this protocol you will miss:
- The fastest way to troubleshoot primary antibody western blot problems that lead to blank film
- The most effective method for finding the best primary antibody
- The best way to get a publishable Western blot for your proteins
Step 2 in western blot.org’s blank film troubleshooting protocol will examine issues related to primary antibody and antigen problems. By now you should at a minimum have good reagents (secondary antibody, western blot markers, blocking buffer, wash buffer, ECL) and know that your HRP-labeled secondary antibody will bind your primary antibody.
To be clear, you should no longer have blank film, because your markers show up every time you blot.
The Fixes – Do these in order the total time needed is X hrs and X minutes, do not skip ahead
To complete this protocol you will need at least 20 loads of your target protein whether its a cell lysate, purified protein, tissue lysate, or whatever. You will also need a sufficient quantity of primary antibody to complete X of blots.
1. Check your primary antibody species reactivity. Total time needed to complete this step 10-30 min.
This step is easy. Make damn sure your primary antibody reacts with the species of your target protein. Most antibody suppliers do a decent job of telling you the antigen the protein was raised against, but don’t trust anyone. Read carefully, do a literature search, pick up the phone and call somebody. If your protein is in CHO cells you need cross reactivity with a hamster. HeLa = human, etc.
Sometimes you will see something that looks like this “H (Mk)” under reactivity for a primary antibody on a manufacturers website or data sheet, where H = human and Mk = monkey. Its critical to note that the parenthesis around (Mk) means hypothetical. Hypothetical as in predicted reactivity based on 100% sequence similarity. Not hypothetical as in – we ran 100 Western blots and the antibody cross reacts with monkey protein of interest.
2. Run a positive control dot blot with the primary antibody. Time needed 2 days of western blot troubleshooting.
This is one of the hardest steps for troubleshooting blank film. You are going to be tempted to skip this step and move on to running the “real” western blot with your sample. In fact, I bet you did it already. Stop it! Bad scientist. Positive controls are your life line for successful Western blots.
The hardest part about positive controls is finding one! But fear not, there’s a method to getting it right.
Source for positive western blot controls
1. Antibody manufacturers. Most antibody providers these days give great information about the antibody and usually include both positive controls and literature citations for Western blots.
In the example image, you will see that this antibody for Caspase-4 was tested in 3 cell different extracts by Western blot – KARPAS-299, THP-1, and IM-9 cells. The image is taken off the Cell Signaling website. Incidentally no where on the page do they mention that these cell lines are a positive control for Caspase-4 antibody, you have to look at the western blot image to figure it out. Its likely that your lab or at least some lab at your university/company is growing these cells. Go get a flask and make a fresh extract.
Otherwise, you might call the manufacturer and get them to send you an extract to test their antibody, or just buy the cell line from the ATCC or other source and make your own extract.
Not enough primary or secondary antibody is bound to the protein of interest.
Use more concentrated antibody. Incubate longer (e.g. overnight) at 4ºC.
Use a mild detergent such as Tween20 or switch blocking reagent (i.e. commonly used blocking reagents are milk, BSA, serum or gelatin).
Check the datasheet or perform a ClustalW alignment to ensure your antibody should react with the target protein; Run the recommended positive control.
Insufficient antigen.
Load at least 20-30 μg protein per lane; Use protease inhibitors; Run the recommended positive control.
The protein of interest is not abundantly present in the tissue.
Use an enrichment step to maximize the signal (e.g. prepare nuclear lysates for a nuclear protein, etc.).
Poor transfer of protein to membrane.
Check the transfer with a reversible stain such as Ponceau S; check that the transfer was not performed the wrong way; if using PVDF membrane make sure you pre-soak the membrane in MeOH then in transfer buffer.
Excessive washing of the membrane.
Do not over wash the membrane.
Too much blocking does not allow you to visualize your protein of interest.
Instead of using 5% milk in the antibody buffers try removing the milk or using 0.5%. Switch blocking
reagents or block for less time.
Over-use of the primary antibody.
Use fresh antibody as the effective concentration is lowered upon each re-use.
Secondary antibody inhibited by sodium azide.
Do not use sodium azide together with HRP-conjugated antibodies.
Use fresh substrate.