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Stripping and Reprobing Problems

Stripping and Reprobing Problems - Western Blot Forum

Stripping and Reprobing Problems - Discuss western blotting and immunoblot in the western blot forum discussion board. Ask Questions about transfers, blocking, membrane antibody incubation and exposures.


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  #1  
Old 06-22-2012, 05:35 PM
Pipette Filler
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Default Stripping and Reprobing Problems



Hi Everyone,

I don't think I've not seen a thread on here about this yet, but if there is, I'm sorry for reposting

My Problem:

My initial probing of the Western Blot appears to go well, I can see that I've gotten pretty good transfer and can see my bands of interest.

When I strip the blot and go to reprobe it, I am not seeing anything beyond background banding. This includes not being able to visualize the ladder.

My thoughts were that I was too aggressive in my stripping and that I wasn't removing enough of the SDS or that my concentrations were too high. But I've double checked it all, have done excessive washes with TONS of TBST followed by TBS before reblocking and I still have this problem.

When I change species (my first blot is rabbit and after strip and reprobe the next set of antibodies is mouse) I get no signal at all, not even background levels.

Any help would be appreciated

Thanks in advance!

JB
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  #2  
Old 06-23-2012, 11:10 PM
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Default Re: Stripping and Reprobing Problems

Please list your stripping buffer recipe or the name and where you purchase it from.

I've used the "Harsh Stripping Buffer" recipe from [Only registered users see links. ] with good results.

Last edited by jeeep; 06-23-2012 at 11:11 PM.
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  #3  
Old 06-25-2012, 01:25 AM
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Default Re: Stripping and Reprobing Problems

The Stripping buffer is
62.5 mM Tris-HCl (pH 6.8)
2% SDS
0.1 M Beta Mercapto
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Old 06-25-2012, 08:33 AM
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Default Re: Stripping and Reprobing Problems

Here my protocol for stripping a western:

(After probing for protein of interest)
-Briefly wash in TBST (1 min) to remove ECL
-Add Stripping Buffer to cover WB, place on shaker for 10min.
-After 10min add ECL and re-expose in imagestaion to confirm stripping
-Wash 4 times, 5 mins each in TBST
-Block 1 hr RT in 5% BSA or o/n at 4C
-Add primary ab, etc.....
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Old 06-25-2012, 04:23 PM
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Default Re: Stripping and Reprobing Problems

Thanks, I'll try that out,

I was rinsing the WB in TBS after probing
Then 30 mins in 50 C bath with stripping
Then 3 x 10 min washes with TBST rinses while shaking
Then a few more rinses with TBS
Then blocking
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Old 06-25-2012, 04:52 PM
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Default Re: Stripping and Reprobing Problems

My guess is that the 50 C, 30 min with stripping buffer is the problem. Just try 10 min then add some ECl and expose it to confirm.
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Old 06-25-2012, 11:07 PM
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Default Re: Stripping and Reprobing Problems

Thanks!
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