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Trouble with Western Blot Transfer (Poor transfer)


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#1 haman

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Posted 17 March 2005 - 11:45 AM

I am doing transfer for 6 months and results are negative.
Coomasie Staining indicates most of the proteins stayed in the gel.
Electrophoresis seems to run nicely and marker gets transferred well to the nitrocellulose membrane, too.
I am using Novex transfer module and doing transfer for 3 hrs with 25V.
Transfer buffer is composed of 48mM Tris Base, 39mM glycine, 0.037%SDS, and 20% methanol in a liter. pH is around 9.25.
The protein what I am interested in is Hsp70 families.
This protocol used to work like a year ago and now I am in trouble.

I am going to try 4 hrs of transfer with 30 V and with Icepack and 10% methanol next time.

Does presoaking gel or membrane in transfer buffer help?

I usually presoak membrane for 5 min and does not presoak gel.

Or could this be the problem of protein isolation/homogenization?
and if the isolation or homogenization or lysis are the cause, how can they affect the transfer?

Please help!
I appreciate your attention.

#2 mabusheh

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Posted 17 March 2005 - 08:10 PM

Hi
what membrane do u use? Nitrocellulose or PVDF, I recommend PVDF of Pore size 0.45. try this if it works. Also, try and do a wet transfer at 350mA
for 4hrs. 20% meOH tansfer buffer seems fine, I myself use the same composition and it works ok.
since the the gel run nicely and the marker transfer ok. then your protien might need more time for transfer.
and also, to be safe enough, soak your gel for 5mins. activate your membran before using with 100% methanol for couple of mins then soak the membrane in transfer buffer for at least 10 mins.
let us know the results once you get them
good luck

Edited by mabusheh, 17 March 2005 - 08:13 PM.


#3 haman

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Posted 17 March 2005 - 11:12 PM

Thank you very much.
I'll let you know the result as soon as I do the next transfer

#4 fred_33

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Posted 18 March 2005 - 01:15 AM

hi
try also without using sds in your transfert buffer... Some discussions on this forum point the fact that sds in transfert buffer is not good...
i 'll put the links later (i need to find them but i prefer tell you that before)...




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