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bad western blot! what is the cause?


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3 replies to this topic

#1 jasmina

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Posted 26 March 2010 - 08:02 AM

Hi everyone,
I made western blot on my membrane protein (MW=114 Kda). the transfert was on semi-dry apparatus. the running on nitrocel. membrane took along time!! almost 3 hours (100V). after ponceau stain, there are bands on the middle and at the bottom of the filter. coomassie stain shows some bands on the gel!!! does it mean that the transfert was partial???
after ECL plus exposure, in the storm, i saw many aspecifics!! faint band , but not at the right MW of my protein.even in the positive control, no band!
In the western i made before, i couldnt see my protein, because the antibody is not working!! the transfer was done in tank blot apparatus, and the ponceau shows good staining of the filter, and coomassie stain was without any bands!!
my experiments are blocked, since without good result on western, i cant go ahead!!
if you know company that deliver a good antibodies, please help
thanks

#2 mdfenko

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Posted 26 March 2010 - 08:21 AM

you can improve the transfer of high molecular weight proteins by adding up to 0.05% sds to the transfer buffer.
talent does what it can
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#3 jasmina

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Posted 29 March 2010 - 03:03 AM

Hi,
thanks for your answer, in fact, my transfer buffer contains glYCINE (29g), Trismabase (58g) and 20% SDS (19.5ml), then, 20% methanol.

#4 mdfenko

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Posted 29 March 2010 - 10:16 AM

20% sds?

that is a lot, the sds can interfere with binding. try reducing to 0.05% sds (leave the methanol at 20% in the buffer).

Edited by mdfenko, 29 March 2010 - 10:16 AM.

talent does what it can
genius does what it must
i do what i get paid to do




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