I am intersted in detecting the p-mTOR protein, which is around 290 kDa. Can anyone give me some tips on how to efficiently blot for proteins in this size-range?
At the moment, I am using 4-20% precast gels using the TGX buffer system (Bio-rad) and loading ~15 ug total protein. Importantly, I'm also doing my transfers with the Trans-turbo system from Bio-Rad.
Some tips you might want to consider:
1) Instead of 4-20% gradient gel I would suggest gels with 7.5% or 10% fixed acrylamide concentrations (the lower acrylamide concentration the better efficiency of transfer)
2) High MW proteins require longer time for transfer from the gel to the membrane (increase the transfer time from 2 h to 4 h or even 6 h)
3) It is better not to increase current of the transfer (because that might result in excessive heating and partial denaturation of your protein with its loosing immunoreactivity to monoclonal antibodies)
4) We did not get much success with Trans-turbo system from Bio-Rad (it may be related with our particular protein). But for such high MW proteins as yours I would prefer to use a regular liquid western blot system in cold room.
Some tips you might want to consider:
1) Instead of 4-20% gradient gel I would suggest gels with 7.5% or 10% fixed acrylamide concentrations (the lower acrylamide concentration the better efficiency of transfer)
2) High MW proteins require longer time for transfer from the gel to the membrane (increase the transfer time from 2 h to 4 h or even 6 h)
3) It is better not to increase current of the transfer (because that might result in excessive heating and partial denaturation of your protein with its loosing immunoreactivity to monoclonal antibodies)
4) We did not get much success with Trans-turbo system from Bio-Rad (it may be related with our particular protein). But for such high MW proteins as yours I would prefer to use a regular liquid western blot system in cold room.
Thanks for the quick feedback, Viktor! We have 7.5% gels in stock so I will give that a go along with the standard transfer tank.
Hey, Khalid!
Viktor already gave you excellent tips.
But I would also try to run the eletrophoresis at a lower current. It will take more time to complete the running, but I think it would help, once bigger proteins take longer to run to the end of the gel.
I use the Bio Rad's Trans-Turbo, and I've never had any problems with it.
Are you selecting the preprogrammed protocol for high MW protein in your equipament? If you are, I also suggest you to try in the stantard transfer tank.
- Do you use nitrocellulose or PVDF membrane? PVDF is more suitable for heigh MW proteins.
Good luck with your blot! ;)
Hello! What is better corrent for big proteins in eletrophoresis and transfer?
Thank you!!!
@ Andre: I have been running the mixed m.w. setting on the Trans-turbo since my loading control is ~50 kDa. So far I have only been working with nitrocellulose but will consider using PVDF. Thanks!
Additional points to consider during transfer---- If you are using Methanol in the buffer for transfer of your proteins, I would suggest using between 0 to 10% methanol. That increases the transfer efficiency. Standard liquid immersion transfer systems work better for large protein transfers, rather than semi-dry systems.
If you do find a good condition that works for you, please update this forum with your conditions.
Thank you
Sanath
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