Western transfer time: Is it crucial for big proteins? - anomaly HELP (Nov/10/2005 )
HI all,
I am going to give a talk next week and will have to explain to my research group on how I have successfully found the protein that I was looking for, migrating at approximately 300 kDa on a 6% gel, that others have had problems detecting. NEED ADVICE!
It is funny that the only alterations I made was that I transferred it after SDS-PAGE for 5 hours instead of overnight and I found this protein. I am also perplexed that previously, people have to use 4% gel and I didn't find anything. Could the protein that was really large, be affected by the gel percentage and the transfer that was previously too long (overnight at 40V) degraded the protein? I was perplexed to see that 5 hours transfer at 70V was sufficient and wondering if transferring it for too long could have caused the protein to pass through the membrane?????? I know they say that proteins that are larger and maybe more hydrophobic may not stick to the membrane as well. maybe previously people washed off the proteins from the membrane without knowing.
sam
u r on the right track..The transfer tme do affect. If you want try and run it in their conditions ....except put another piece of PVDF underneath the first membrane. If the protein is going thru hopefully the inderneath membrane will catch it. You will have ur proof.
Good luck.
Having too long a transfer time can cause the protein to transfer right off the membrane. I don't think degradation or washing is an issue.
I am going to give a talk next week and will have to explain to my research group on how I have successfully found the protein that I was looking for, migrating at approximately 300 kDa on a 6% gel, that others have had problems detecting. NEED ADVICE!
It is funny that the only alterations I made was that I transferred it after SDS-PAGE for 5 hours instead of overnight and I found this protein. I am also perplexed that previously, people have to use 4% gel and I didn't find anything. Could the protein that was really large, be affected by the gel percentage and the transfer that was previously too long (overnight at 40V) degraded the protein? I was perplexed to see that 5 hours transfer at 70V was sufficient and wondering if transferring it for too long could have caused the protein to pass through the membrane?????? I know they say that proteins that are larger and maybe more hydrophobic may not stick to the membrane as well. maybe previously people washed off the proteins from the membrane without knowing.
sam