5 replies to this topic

[Devin Paine]

Hi there;

I have undertaken a thesis on the presence of telomerase in cells treated with various phtoestrogens. I have had trouble in performing the western blot, as I have yet to see any bands appear at the MW telomerase should be present at (approx. 130kd). I have tried lowering the gel concentration to 8% with no luck and I am next considering switching from a semi-dry transfer to a wet transfer.

Just curious to see what everyone thinks the problem may be and whether I am on the right track. Also any wet transfer tips, like time, Volts and milliamp settings would be much appreciated.

Thanks.

[bob1]

For a 130 kDa protein, I would be running a 6-7% gel.  Telomerase is quite low abundance IIRC, you may need to be loading more lysate or a more concentrated lysate.

Wet transfer at 15-30 V overnight works well for me.  You can add up to 20% methanol and 0.5% SDS to improve the transfer of large proteins.

[mdfenko]

bob1, on 17 November 2011 - 04:46 PM, said:

You can add up to 20% methanol and 0.5% SDS to improve the transfer of large proteins.

did you mean 0.05% sds?

[bob1]

Whoops, yeah.  I must learn to proof-read, I must learn to proof-read....

[acquire]

For higher size proteins you should decrese 20% Methanol conc to 5-10%
in western transfer( for PVDF membrane.)

Edited by acquire, 05 December 2011 - 05:09 PM.

[mdfenko]

acquire, on 05 December 2011 - 05:08 PM, said:

For higher size proteins you should decrese 20% Methanol conc to 5-10%
in western transfer( for PVDF membrane.)

not necessarily. if you use sds then the higher methanol concentration may be necessary to strip the sds off the protein. it is not membrane type dependent.