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uneven transfer of western blot

uneven transfer of western blot - Protocols and Methods Forum

uneven transfer of western blot - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 08-26-2003, 07:34 PM
Jayakumar, R
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Default uneven transfer of western blot



hi..
How do I determine that the proteins that I find on my coumassie blue stained mini-gel after western transfer is due to protein precipitation or just due to insufficient transfer time? By the way, I still find my proteins on my gel as clear neat bands even after an overnight transfer at 40V, though I should say that atleast 50% of the protein do get transferred even though very poorly at different parts of the gel.
I guess it is embarassing.. but I am not sure how to distinguish precipitated proteins in a 15% gel from that of poorly transferred proteins. Please help.
thanks for all the tips on my previous question.
Jai

---
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  #2  
Old 08-26-2003, 10:55 PM
EK
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Default uneven transfer of western blot

The only way to distinguish is obviously to transfer longer. Howeve, it is
very usual that the transfer is not complete, and that lower MW proteins
migrate faster that higher MW.
I guess Pierce (no affil.) offers an in-gel Western detection system, so
that you don't need to do transfer.
Emir

""Jayakumar, R"" <[Only registered users see links. ]> wrote in message
news:97101976F8A044468CA74FE11883B90E02048912@VIST A.roswellpark.org...
hi..
How do I determine that the proteins that I find on my coumassie blue
stained mini-gel after western transfer is due to protein precipitation or
just due to insufficient transfer time? By the way, I still find my
proteins on my gel as clear neat bands even after an overnight transfer at
40V, though I should say that atleast 50% of the protein do get transferred
even though very poorly at different parts of the gel.
I guess it is embarassing.. but I am not sure how to distinguish
precipitated proteins in a 15% gel from that of poorly transferred proteins.
Please help.
thanks for all the tips on my previous question.
Jai

---


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  #3  
Old 08-31-2003, 04:27 AM
Dr Engelbert Buxbaum
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Default RE: uneven transfer of western blot

"Jayakumar, R" wrote:



In a tank blotter at 40 V even large proteins (150 kDa) transfer in less
than two hours. If not, something is wrong. Tank blotting works better
than semi-dry, btw.

Do not use methanol in your blotting buffer unless you want to transfer
small proteins (less than 20 kDa or so). For large proteins, use 50 uM
SDS in the blotting buffer. Dunn's buffer (10 mM NaHCO3, 3 mM Na2CO3)
works better than Towbin's and is a lot cheaper.

Equilibrate your gel with blotting buffer for 15-30 min.

Make sure that you have no air trapped in your sandwich. Mount blotting
membrane, gel and filter paper with some blotting buffer in a little
tray.

Make sure the sandwich faces the right way (with the membrane towards
the positive pole for western blotting).

During transfer, keep temperature low. Most tanks come with some cooling
system, like ice block or water circulation.

Establish the time required for blotting with a series of blotts for,
say, 30, 60, 90... min, until the gel no longer stains with CBB.
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