Stripping for Science! (…Your membrane of course)
Why would you strip your western blot membrane?
Western blot stripping has the advantages of saving resources, materials and time.
The cost of performing a western blot can build up if resources are used frequently, and it may indeed be more economical and scientifically more beneficial to produce a stripping buffer and re-probe your western blot membrane, rather than starting a new one.
Stripping can be a useful technique for the purpose of:
- Comparing the relative abundance of two proteins.
- Detecting Proteins of similar molecular weights.
- Analysing loading control proteins.
- Confirmation of the result, using an alternative antibody or same antibody as a repeat measure.
When should you strip your membrane?
The process is carried out as a post-detection step, and if your research requires analysis of multiple proteins within the same expression system this could be a valuable tool. In circumstances where a secondary protein is of a similar molecular mass to the initial target protein cutting and cropping the membrane is unlikely to yield the result you are looking for. Membrane stripping will allow for a more wholesome analysis.
Not only can membrane stripping provide further analysis on your western blot data, but it can also be beneficial in circumstances like:
- Removing antibodies which were incorrectly used.
- Modifying and optimising the appropriate antibody concentration
- Reapplying blocking buffer due to high background
Does the type of Western Blot membrane make a difference?
Yes. If you think you may need to or want to strip your antibodies from your membrane, you may want to consider the type of membrane you have used during your experiment.
The two most common western blot membranes are; Polyvinylidene Difluoride membrane (PVDF) and Nitrocellulose membrane (NC). Nitrocellulose is not recommended for re-probing after use as portions of protein signal are known to be lost along with the primary and secondary antibodies.
PVDF, in contrast, is much better at retaining the signal and Western Blot stripping does not damage the proteins embedded within the membrane.
How to strip! (your membrane)
There are two common ways to strip your membrane; a mild stripping which isn’t too intense and then a harsh stripping protocol. Below we've given you an outline for each type. Depending on your requirements you may choose one or the other.
Mild Western Blot Stripping Protocol: (The low pH glycine solution method)
Set up a solution to bring about a final solution with:
- 1.5% (w/v) Glycine;
- Between 0.1-1% (w/v) SDS;
- 1% (v/v) Tween20 in Deionized water;
- Adjust to pH 2.2
The Western Blot membrane is then soaked in the stripping buffer for 20 minutes. At the 10 minute mark, remove the buffer and apply fresh stripping buffer. Following this, rinse the membrane twice with PSB for 10 minutes, then twice again with PBS but for 5 minutes.
The Glycine solution helps to dissociate the bound antibodies, whilst the low pH alters the structure of the bound antibodies so that their binding site structures are altered and rendered inactive (deactivated protein regions).
Harsh Western Blot Stripping Protocol:
Prepare a final solution in deionized water containing:
- 1% SDS
- 62.5mM Tris HCl, pH 6.8
- 0.8% beta-mercaptoethanol
Heat the buffer at 50oC and agitate the Western Blot membrane slightly for 30-45 minutes. You’ll need to rinse for a while with running water to remove any excess stripping buffer. Any remaining beta-mercaptoethanol will denature the fresh batch of antibodies applied and neither of us want that to happen. Then carry out a number of catch washes using TBST so that the membrane is ready to block again.
Did the Membrane Stripping work?
You’ve made it this far and now it’s time to check if the membrane stripping was successful. To do this, first block with the appropriate blocking agent and then follow with the secondary antibody. If the primary antibody was successfully removed, no signal bands should be detected by your secondary antibodies. At this stage either the membrane will be clean, in which case the western blot can be washed and you can apply your primary antibodies or, if there are bands then your strip wasn’t completely successful and you’ll need to repeat the procedure to ensure all of the antibodies are gone from the transfer membrane.
It could be quite distressing to find out after conducting a stripping procedure that it was not effective in removing all antibodies the first time, so we recommend testing your membrane stripping skills by using on a negative control first. When you are confident you could then go ahead and strip your actual western blot membrane.