Western blotting uses electrophoresis and antibody-epitope affinity to give a semi-quantitative and (theoretically) clear measure of protein abundance. It’s a long procedure, filled with many steps—and even more room for error. Learning to troubleshoot certain problems is incredibly important for continued success with this technique.
So what do you do when your final imaged product has astonishingly little signal? The rectangle of nothingness strikes everyone at some point, and there are several places where things could have gone wrong.
Let’s break it down step by step.
Step 1: Gel Electrophoresis
This is what separates the proteins from the… well, proteins. Let’s assume you’ve prepared the samples correctly, and have pipetted them into the wells.
- If your protein of interest is small, you risk running it off the gel. Keep track of where your protein is during electrophoresis by using a visible protein ladder, and be generally aware that smaller proteins will finish faster.
- Be sure to use an appropriately size/percentage gel and an acceptable buffer!
- Match the colors correctly. Turning on the current can seem like a mindless task, but if the electrodes are applied backwards, you’ll get nothing but a headache. This applies to transfer as well. Red to red, and double check!
Step 2: Transfer
Getting the protein to the membrane is necessary for antibody binding—mistakes here will make future steps worthless. Consider:
- Transfer time: over or under-transferring significantly will leave little to no protein for the primary antibody to bind. Like with electrophoresis, smaller proteins move faster! To test for over-transfer, stick a second membrane behind the first and stain it for total protein.
- Buffer: an inappropriate transfer buffer will prevent protein migration to the membrane. Check that your current is appropriate given your constant voltage (or vice versa).
- Membrane activation: this step, required for PVDF membranes, is performed prior to beginning transfer. It’s necessary to make the gel’s proteins “stick.”
Step 3: Primary Antibody
If the primary antibody doesn’t bind where it should (your target), neither will the secondary. Two questions to ask yourself:
- Has your protein of interest been documented (in the literature or by your lab) in your sample type? Proteins in muscle may not be found in serum, for example. If not, your first step should be a titration blot to determine if and/or at what concentration you can get a clear signal.
- Does the data sheet mention that it is suitable for western blots? Some antibodies are only for other uses. Load a positive control!
Step 4: Secondary Antibody
The link between primary binding and imaging, secondary incubation is both simple and incredibly important.
- Make sure the species and isotype matches the primary antibody! Another seemingly mindless task, matching the antibodies sometimes gets overlooked.
- Ensure that your secondary is compatible (conjugated) with your method of imaging in mind—westerns and immunofluorescence often use different types of antibody.
Step 5: Imaging
Often viewed as a black box where membranes go in and pictures come out, be aware that understanding basic imager manipulation is almost as important as getting the technique right in the first place!
- Chemicals: be sure your reagents and protocols mix—you may not do the same thing as your imager-abusing neighbor if you are using film.
- Exposure time: if the automatic settings aren’t producing what you want, manually set it up to go for a longer period of time. Play with signal accumulation modes to compare pictures as you go.
- Contrast and image settings: your image may look bleak, but adjusting the brightness, contrast, and other settings can sometimes illuminate a faint band.
- Chemical agents: sometimes using a stronger imaging agent, such as Thermo’s Super Signal with a longer incubation time, can help clear up your bands and require a shorter imaging time.
Until Next Time…
Although the rectangle of nothingness does happen, if the steps above are addressed, you should always get some signal.
In the next installment of the series, we’ll address the opposite problem: non-specific binding and high background signal.