re-probing membrane - (Apr/27/2007 )

Hi again!
Just a quick question concerning the re-probing of western blot membranes: I use nitrocellulose membranes. If I want to re-probe with another antibody do I need to strip it first or it is not necessary. I tried re-probing a membrane (which was dry and I started out with blocking solution) without striping first and got absolutely nothing at the end Was it because I didn't strip? If so, any good protocols for striping? I'm trying to detect GPCR.

Thanks!!!

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I advice stripping if...

re-probing will be performed with the same species-specific 2nd Ab

or

if re-probed target runs on the same molecular size as probed target

or

if any Ab (for probing or re-probing) exhibits some cross binding reactions


as stripping, f.i. with commercial reagents such as Restore from Pierece, lasts only 15 min, there is no good reason NOT to strip...

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Hi,

I agree with Bearer, strip your membrane only when it is necessary. I happily re-probed membranes (30 ugve/well loaded) 3-4 times without stripping and even more 6-7 times with 2 stripping. My experience is that home-made stripping buffer used at 50C waterbath agitating for 25-30 min results in a significant loss of proteins and sometimes washes off my little amount of target protein. I usually try and plan re-probing and stripping starting with the most sensitive ones (either because of low abundance or poorly performing ab) and strip as few as possible. I also do a loading control after last stripping (no matter if I did it before in one of the re-probing steps), because again, I experienced that sometimes the lanes at the edge loose more protein (probably due to unadequate prodecure).

I've also tried a commercial kit (probably chemicon, I'm not sure) - it was good and very convenient, I wish I was allowed to use it all the time

One more thing: I usually use PVDF membrane, probably that matters, too.
As far as I know, it is not a problem with nitrocellulose if it dries out, but maybe worth checking.
Also, can you detect your protein easily if you do it as first probe? If you don't have nice strong bands for the first probe, stripping can make it even worse.
Are you sure, the transform is OK (semi-dry can be tricky) and you have your protein on the blot?

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Hi!

Bearer:
Thanks for the advice. I'm rebrobing with the same species specific 2nd Ab but the reprobe target runs at a different molecular size than the probed target (what is the problem with using the same 2nd Ab?). I cannot be sure if there is cross binding reactions concerning the Ab for probe or reprobe target but there isn't supposed to be any.
I will try again without stripping in case something else went wrong the first time and then with stripping if I get nothing again! Thanks again.

Krisztina:
Thank you too! It is true that I'm a bit scared of loosing some of my protein of interest taking in account that it is probably not a lot. That is way I didn't strip in the first place.
Sorry, but I'm a bit new at this...what is a loading control?
I was told that there isn't a problem with nitrocellulose when it dries as long as somebody starts with the blocking solution when reprobing so that the membrane is wet when going for the primary Ab.
I haven't tried a first probe for the specific protein...I tried saving some time to check if I can find that protein by reprobing a membrane I had probed for another protein. Anyway I will do a first probe for that protein for sure but I was just wondering if I could have a "quick look" before going for the full procedure!
Thanks for you tips!

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Hi Charis,

1. The loading control is to make sure the differences you see on your blot are not because of unequal loading of the wells but because of real changes upon treatment. Threfore you want to show the equal loading of your gel. All reviewers are likely to ask for it in order to believe your data. Easiest way: Ponceau-staining and making a picture of it. I do not recommend it, though, since it is not easy to make a good image of you P-stained blot before the bands fade.
Usual way: try and find any protein that is not likely to change in yr experiments (GAPDH, actin, tubulin - these are common ones but often you need another one, anything that you have antibody for and you checked it doesn't change (do Ponceau and make sure loading is equal and probe your membrane to see it is equal, too).
2. When you use the same sec ab, it is likely that you'll see the bands from the previous probing, too. It is usually not a big problem, if you have only one band for each probe far away from one another. But if they are close or the probe gives multiple bands, it is a problem. In that case, plan your probes smartly, for instance rabbit-HRP, mouse-HRP, strip and rabbit again. In this way you can make a better use of your membrane.
3. My knowloedge is the same on nitrocellulose.

BEst wishes!

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Hi Krisztina!
Thanks for your time and tips, appreciate it !

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Good luck with your experiments, Charis, I wish you nice and clean blots with strong bands!

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