We've all been there: You're at the end of your experiment and your Western blot protocol — the one that worked 24 hours earlier — is suddenly giving you fits. Try as you might, you just can't replicate your results and get that publication quality image. It's time to start troubleshooting. But where do you start?
Here are some possible issues (and fixes) for when you don't see any bands on your blot:
1) Poor lysate preparation
A lack of signal often results from improper lysate preparation or insufficient protein concentration. For over-concentrated or "dirty" samples, try titering the lysate until you get a better signal.
Increase the concentration of your primary and/or secondary antibodies (using freshly prepared dilution), referencing the product data sheets for recommended dilutions.
Check the date on your lysis buffer. Always use fresh reagents to ensure proper disruption of the cell membrane.
2) Improper gel
Large proteins should be run on lower percentage gels and transferred overnight at 4°C, with SDS in the buffer. With small proteins, opt for a membrane with smaller pore size, such as 0.2 um.
Some antibodies will not bind to the denatured form of the protein; consider a "native" or non-denaturing gel.
3) Wrong antibody
Confirm the antibody's species reactivity on the product site or on the product datasheet. Check serial and batch numbers to make sure you're using your intended product. Many have similar names or abbreviations.
4) Non-specific binding
Non-specific binding occurs when antibody concentration is too high or the antibody is "dirty", meaning it recognizes proteins besides the target of interest. Another possibility is that the antibody is binding proteins that have had high affinity binding sites exposed during lysis.
The easiest way to remedy the problem is to extend the blocking step prior to the first incubation. Blocking is most often performed with BSA or dried milk in TBS-T, both of which contain a mix of natural proteins. Incubating for an hour in one of these solutions will occupy any high-affinity sites on your membrane that may otherwise bind your primary antibody and provide a false signal.
To test non-specific binding of the secondary antibody, consider running a second gel under identical conditions, but omit the primary antibody during the first incubation step.
5) Failed transfer
Examine the condition of your transfer cassettes for any broken hinges or connections. Uneven pressure across the transfer "sandwich" from a broken hinge can lead to uneven signal levels across the membrane during developing.
Confirm that all electrical connections to your transfer tank are properly aligned and free from significant wear or corrosion.
Perform a Ponceau stain after the transfer step. If protein is present but no signal is recorded, check antibody stocks and concentrations. If no protein is detected, consider increasing the loading weight of your lysate or adjusting the gel composition or transfer conditions.
6) Burnt out signal
Check if there is extra ECL (or other luminescent substrate) remaining on or around your membrane or in your developing cassette before inserting the film. Holding the corner of membrane with tweezers, gently shake to remove excess liquid. Carefully remove any remaining substrate from the casette with a kimwipe or paper towel, taking care not to touch the membrane directly.
If you observe white bands (possibly surrounded by black) where your protein of interest is expected, it's possible your protein concentration is too high, resulting in a quick "burn out" of your ECL. Titer the protein lysate and and dilute your antibodies to recover a signal.
7) ECL or development
Mix your ECL reagents fresh each time you develop. If no signal is visible at first, increase the exposure time. While optimal exposure will usually be somewhere between 1 and 10 minutes, certain protocols may require 15, 30, or even 60 minutes. Just make sure to keep the cassette in a dark location, such as a drawer or heavy plastic bag, if you decide to leave your developing area, since even the smallest bit of light penetration during a long exposure can lead to an unusable film.