I am planning western blots using different lectins to test the glycosylation of some samples. As the material is often available in limited quantity (e.g. patient material) it would be great to re-use the membrane after stripping and test with another lectin. This is done quite often with antibodies but I could not find enough information in the web for lectins.
Hi Chantal, there are numerous ways to strip a membrane for re-probing. You can use 1% SDS, 0.1M acetic acid pH 3, 8M urea, etc.
Before using your patient samples, you probably should do a test of running plain serum on a gel, blot it and see what stripping method works best.
Also, Vector has good biotinylated lectin kits that you can test.
https://www.vectorlabs.com/catalog.aspx?catID=59
Thank you for the answer. I already did some stripping to test different antibodies on samples and it worked fine. I was wondering if someone did it already with lectins and had some experience with it, as lectins are different to antibodies... Anyway I am going to test my lectins on cell lines first and look if it works ;) My lectins are actually from Vector as I had already good experience with them :).
Dear Chantal, we already tried to strip nitrocellulose membranes that where incubated with lectins. Unfortunately we weren't able to strip them efficiently, yet. When we did this after incubation with an antibody, it actually worked quite well. If you can manage to strip your membrane, it would be awesome to share which buffer you used.
Hi Chantal, I was able to strip nitrocellulose membrane that was incubated with a lectin that I work with. However, my second blot was done with an antibody (not a second lectin) but maybe you could try the same approach with PVDF membrane.
I work with a lectin from jackfruit seed that has affinity for Mannose, so I used abcam mild stripping protocol followed by 4 washes at room temperature (15min each) with TBST containing 100mM Mannose. Afterwards I just washed the membranes with TBST (3x10min), reblocked with BSA (the whole procedure was done with BSA and not milk) and probed with the antibody that I needed. I had a good result considering that my lectins binds to HRP, so I was expecting that any remaining lectin would leave a huge background after ECL exposure.
Hi Eleonora, I have been trying several protocol for stripping and they did not work for my samples. Anyway, the lectin blot worked well. Here is my protocol for PVDF membranes:
-Blocking 1h @RT shaking gently with Odyssey Blocking Buffer (TBS)
- Incubation with 10 ml Lectin Solution: 10 µL lectin of choice (biotin conjugate), 10 ml Odyssey Blocking Buffer (TBS), 20µl Tween 20 O/N shaking @4°C
- Washing: 5x5min with TBST
- Incubation with 10 ml streptavidin (conjugate of choice) solution: 5 µL streptavidin , 10 ml Odyssey Blocking Buffer (TBS), 20µl Tween 20 shaking @RT for 1h
- Washing: 5x5min with TBST, 2x5min with TBS
What kind of problems are you having?
Best, Chantal
In general it is very important that you do not use any reagents (blocking buffer etc.), which contain glycosylated proteins!
Best, Sandra
A related question:
If I want to analyse the lectin binding and the protein amount of ONE protein (= ONE band) in western blot, the best way to do it would clearly be just running two gels and doing two blots. But also my sample amount is limited.
First lectin detection and the antibody detection seems to be difficult, because it is hard to strip lectins.
Has anyone tried the opposite? Antibody detection, stripping, and then lectin detection?
Best wishes, Jan
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