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Western Blot for 320KD Protein

How to optimise

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4 replies to this topic

#1 bochum

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Posted 27 September 2011 - 09:01 AM

For my work i have to do a western blott for 320 KD protein. I tried 7% gel. Bought a gradient gel of 2%-16%. Run the gel overnight (4 degree). Transferred 3hr in cold room. Still not able to detect the protein.

Suggest where am failing.

Thanks
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#2 casandra

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Posted 27 September 2011 - 09:10 AM

For my work i have to do a western blott for 320 KD protein. I tried 7% gel. Bought a gradient gel of 2%-16%. Run the gel overnight (4 degree). Transferred 3hr in cold room. Still not able to detect the protein.

Suggest where am failing.

Thanks

do you have a positive control for your antibody or your western blotting technique as a whole?
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#3 bob1

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Posted 27 September 2011 - 04:10 PM

Large proteins, because they are typically low abundance (the cell can't produce much of them because of the size), require that you load a lot (try titrating the amount) of protein to detect them.

#4 mdfenko

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Posted 28 September 2011 - 07:29 AM

did you add 0.05% sds and 10-20% methanol to the transfer buffer?

the sds will assist in getting the protein out of the gel and the methanol will strip off the sds.
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#5 LifeTein Peptide

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Posted 19 October 2011 - 09:26 AM

Here is a useful protocol for big protein like this 320 KD and small proteins such as 30KD. See links below or the attachment.

http://www.lifetein....ll_peptide.html
http://www.nature.co...prot.2006.4.pdf

Attached Files


LifeTein, The Peptide Company
www.lifetein.com
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