I was wondering, and didn't find so much on the web, if there was a stripping method for nitrocellulose membranes, to get rid of my previous stains.
In our lab we use (I think the most common) stripping protocol of (vol/vol):
- TRIS pH6.7 - 62.5%;
- SDS 20% - 20%;
- ß-mercapto-ETOH - 0.7%
- H2O to 100%
- 50°C - 65°C for 30-45mins, agitate repeatedly
With a lot of stains that works out fine, but with some more highly expressed proteins stained with "good" ABs (let's say ERK1/2, CS), the signal persists after stripping. I also tweaked it a bit by doubling or tripling the mercapto, longer heating, but with little success.
I am not afraid of losing protein - if that happens I can still soften the method. So I'd be thankful for any suggestions (short of sandpaper) how to get rid of these ABs.
Cheers!
I have a protocol for PVDF membrane but I am not sure if this is good for Nitrocellulose membrane.
1: Glycin 15 g
2: SDS 1 g
3: 10 ml Tween 20
4: Adjust the pH to 2.2 with HCl and ad water to 1 l.
After the first blot, if the membrane is not dried up, than start the following process:
1: Two ten min wash with the stripping buffer
2: One wash of ten min with the TBST buffer to bring back the pH to 7.5
3: Reactivate the membrane by methanol (2 min incubation)
3: Repeat step 2
4: Block it for half an hour
5: Incubate with the primary antibody overnight.
I am not sure if this works with nitrocellulose membrane but you can search on net for anything like this with a modification for NCM.
All the best.
I have had good experiences with the Restore reagent from Pierce Scientific, though I am not sure if it would be "stringent" enough for what you are seeking to achieve.
I have used 400mM Glycine at pH 2.5 to strip westerns on nitrocellulose. Shake the western in Glycine for 15-30 mins, wash a couple of times with TBST and then proceed to blocking step of western. It works excellent. I always sterile filter the solution and keep it always at 4C until use. Good luck!!
I have used Re-Blot Plus Strong Solution from Millipore Catalog Number:2504 It work well to remove alpha tubulin which is highly expressed in my cells and hard to remove. So you can try this after diluting 10 times. Usually they recommend 5 minutes but I have tried upto 15 minutes with this. After stripping I usually ECL the blot to see if band is gone for some tough band as it is a kind of assurance before you start blotting for next antibodies. I hope it help.
@Edgardo: Thanks, that sounds simple enough, I am just trying it right now. I also found (actually on the Millipore site) that people use glycine 25mM + 1% SDS at pH 2 - I will also try that.
@Daleep: Thanks, I will try the Millipore agent in case the glycine low PH stripping doesn't work.
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