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WB for large proteins - Transfer conditions with PVDF membranes (Jul/22/2005 )

Fellows,

I'd like to know your opinion on protein transfer conditions into PVDF membranes for large proteins, let's say over 100 KDa. I'm working with a couple of proteins (94 & 116 KDa) and my results have been very dissapointing so far (too few transfered protein, too much retained on gels). I'm using a transfer buffer with 20% methanol, and transfer settings are 100-120V for 90-120 minutes.

Any suggestions? All comments will be very appreciated. Thanks.

-Carbio-

QUOTE (Carbio @ Jul 22 2005, 10:47 AM)
Fellows,

I'd like to know your opinion on protein transfer conditions into PVDF membranes for large proteins, let's say over 100 KDa. I'm working with a couple of proteins (94 & 116 KDa) and my results have been very dissapointing so far (too few transfered protein, too much retained on gels). I'm using a transfer buffer with 20% methanol, and transfer settings are 100-120V for 90-120 minutes.

Any suggestions? All comments will be very appreciated. Thanks.


A few more details would be helpful, % of gel, your transfer buffer concentrations would be very helpful.

The mass of your protein is not large at all. I routinely transfer 100kDa proteins w/out a problem, and the 250kDa standard transfers all the time as well.

It might be possible that the the ionic strength of your buffer is very weak, if it is, then that would explain possibly your observations. But the issue isn't the mass of your protein. Large proteins are generally considered to be 1 MDa, like Ahnak, that's a large protein

-viper-

the parameters you used for protein transfer have no problem for me.
Could you tell me how about low is the transfer efficiency of low molecular weight markers? Fully transferred?
Since your protein is partially transferred, I feel there's something wrong with your transfer buffer. Please check it, make sure no mistakes during buffer preparing.

-bullfrog-

hi
regarding your buffer recipe, some persons use only 5% methanol. And i agree with the first poster of this topic, a i've heared too that too ethanol reduces high MW proteins tranfert.
Btw, i don't use methanol for my tranferts (nitrocellulose membrane) and i can great transfert up to 150 kDa...
fred

-fred_33-

Greetings! I'm in a high through-put Western blotting lab, and we use only 10% methanol in our transfer buffer, and no SDS. You should have no problems with this buffer. Perhaps you could increase your transfer time and/or amperage. We routinely run Western blots on proteins above 100kDa in weight with good reproducible results.
Good Luck!
monkeybaji

-monkeybaji-

hi,
i have transferred 300 and 560kda proteins routinely. it always helps to have 5-10%methanol, 0.1% SDS and longer transfer (3-4)hours. of course for my proteins i have to do overnight. keep the power at 75v.
good luck
sat

-biosat-