Protein samples not transferring over to membrane - Western Blot (Oct/03/2006 )
If you think your 18kd protein is being transferred through your nitrocellulose (smaller proteins can do this) you can put 2 nitrocellulose filters and check the "second" one for transfer. Good luck
quote name='whatthe' date='Oct 5 2006, 02:48 PM' post='71929']
The protein which I am having trouble transferring is actually very small, around 18kd. But initially I was having trouble transferring over even the larger proteins and currently I have solved that problem by extending the transfer time. I did it at 100V at room temperature for an hour. Also, be sure you add SDS to your transfer buffer, I use 0.04% but up to 0.1% can be used. I tried it without SDS before because I was worried my small protein would pass through the membrane and didn't get a transfer at all! I still haven't gotten my smaller proteins to transfer but for a large protein like yours, increasing transfer time should work.
About the diffusion problem, I was thinking perhaps soaking my gel after I ran the sds page and before i do my transfer would have caused the smaller proteins to diffuse out? I heard for small proteins we should not soak the gel in transfer buffer for such a long time, esp. since I am using a mini-gel. I had soaked my gel for quite a bit like 20 minutes so there's a chance the proteins could have diffused do you think?
mdfenko... I do use a 0.2um pore but thanks for the suggestion!
is yrs a veryy basic protein.. basic proteins may have problems with transfers! after checking whether it has run thru or what.. using double membrane.. u can try using methanol or something in the transfer buffer. g'luck!!
i regulary transfer proteins onto NCM of a size of 13kda. if i run for any longer than 15 mins at 13V on a biorad semi dry all the proteins run through and will not show up.
oh and also i always soak my gel in the transfer buffer for my small proteins for as long as half hour once, and have not had any problems