Membrane stripping for western blot

Essential guidance for removing antibodies from western blot membranes.

What is membrane stripping?

Membrane stripping is the process of removing primary and secondary antibodies from a western blot membrane. Its purpose is to allow the investigation of multiple proteins on the same blot without cutting the membrane. Membrane stripping is particularly useful when examining a protein of interest alongside a suitable loading control or when studying two proteins of similar molecular weight.

There are alternative scenarios where membrane stripping proves beneficial. For instance, it's helpful when an incorrect antibody has been used or when membrane blocking must be repeated due to high background signals. By stripping and re-probing a single membrane, you can save samples and time, avoiding the need to run and blot multiple gels.

Avoid making quantitative comparisons of targets probed before and after stripping since the procedure removes some sample protein from the membrane. For the same reason, a stripped membrane should not be probed to demonstrate the absence of a protein, like for knock-out testing or an RNAi experiment. We recommend using a PVDF membrane to minimize the loss of sample protein, as nitrocellulose membranes tend to have a more significant protein loss.

Membrane stripping protocols

In the table below, we outline two stripping protocols that differ in their treatment's harshness. As a general rule, try the mild stripping first and then proceed to the harsh one if the signal from the targeted antibody persists.

These steps can be repeated for probing with several antibodies. However, keep in mind that after each stripping round, the potential signal may become weaker, and the background could increase.

Mild stripping

Harsh stripping

Buffer

Buffer, 1L:

Prepare buffer and strip membranes under a fume hood.

15 g glycine

1 g SDS

Buffer, 0.1 L:

10 mL Tween 20

20 mL SDS 10%

Dissolve in 800 mL distilled water

12.5 mL Tris HCl, pH 6.8, 0.5 M

Adjust pH to 2.2

67.5 mL distilled water

Bring volume up to 1 L with distilled water

Add 0.8 mL ß-mercaptoethanol under the fume hood

Procedure

1. Use buffer volume that will cover the membrane and incubate at room temperature for 5–10 min.

1. Warm the buffer to 50°C

2. Discard the buffer

2. Add the buffer to a small plastic box with a tight lid; use a buffer volume that will cover the membrane

3. Repeat incubation for 5–10 min with fresh stripping buffer

3. Add the membrane. Incubate at 50°C for up to 45 min with some agitation

4. Discard the buffer

4. Dispose of the solution as required for ß-mercaptoethanol based buffers

5. Wash for 10 min in PBS x 2 times

5. Rinse the membrane under a running water tap for 1–2 min

6. Wash for 5 min in TBST x 2 times

6. Traces of ß-mercaptoethanol will damage the antibodies. Wash extensively for 5 min in TBST

7. Ready for blocking

Determining the membrane stripping efficiency

To determine the membrane stripping efficiency, you can incubate the membrane with a chemiluminescent detection reagent after the stripping procedure. Avoid using colorimetric/chromogenic detection reagents, as they leave a permanent visible stain on the membrane, which can interfere with the subsequent detection of targets with similar molecular weights. Chemiluminescent reagents like ECL are preferable since they are more sensitive than colorimetric reagents and won't leave any stain.

The most reliable way to test the success of stripping is by re-blocking the membrane and then using a secondary antibody. No signal bands should be detected if the primary antibody was successfully removed. If the membrane is clean, you can wash it to remove any residual secondary antibodies before starting the next primary antibody incubation. If signals still appear, you may need to either escalate from mild to harsh stripping or repeat the entire procedure.

Lastly, it is crucial to rinse the membrane thoroughly with buffer after achieving satisfactory stripping efficiency. The residual stripping buffer should be completely removed, as any leftover may interfere with the follow-up immunodetection process.