7 replies to this topic

[hmohamma]

Hi everybody,

I am having difficulty with transferring proteins from non reducing SDS-PAGE gel to PVDF membrane.
I stain my gels after transfer and the proteins are separated well enough but still there. Plus using Ponceaue red I don't see anything on teh memberane. My pre-stained marker is transferred perfectly. The only concern I have is the cell lysate. It has two fractions: one just a liquid form and the other with very high viscosity. I try to mix them well and take from both parts before running, then I just warm it at 90 for 5 min. I was wondering is it difficult to transfer the proteins in non reducing form. I had couple of times of good transfer but most of the time no luck! Any help is greatly appreciated.

Thank you guys in advance  

Hak

[Prep!]

Hi hak its a common problem and u need not worry a lot abt it... it will be miore helpful if u can give some more details like mol weight of the excpected protein... wb conditions.. buffers.. etc!!

Good luck!!

[hmohamma]

Hi Pradeep Iyer,

Thank you for your response. The proteins I am looking are from 24KDa-120Kda in the cell lysate of virus infected cells. Buffer I am using for transfer has Tris-Glycine in Meth-OH and H2O. So, what I am goiong to do is to run some of my samples in the reducing condition and see how it will transfer. For, people are doing it in the lab with no problem of transferring. These are what I will do. Plus I heard about CHAPS buffer fir transferr but I don't know what it is.

Thank you again

Hak

Pradeep Iyer, on Mar 24 2010, 08:41 PM, said:

Hi hak its a common problem and u need not worry a lot abt it... it will be miore helpful if u can give some more details like mol weight of the excpected protein... wb conditions.. buffers.. etc!!

Good luck!!

[Prep!]

the most basic thing if the transfer efficiency is not proper is to add 0.01 - 0.1% SDS in the transfer buffer or if the pI value is high i.e more than 8.3 which is the transfer buffer pI u might have to try other buffers... but most often than not SDS shud work

Best luck!!

[hmohamma]

Pradeep Iyer
Thank you for the suggestion. Actually that was the subject of my discussion with my advisor today to use SDS! Thanks again.

Hak

Pradeep Iyer, on Mar 25 2010, 09:13 PM, said:

the most basic thing if the transfer efficiency is not proper is to add 0.01 - 0.1% SDS in the transfer buffer or if the pI value is high i.e more than 8.3 which is the transfer buffer pI u might have to try other buffers... but most often than not SDS shud work

Best luck!!

[Prep!]

Oh tats great Hak... do tell us the results of your experimentation!!!!  

[mdfenko]

i agree with pi that sds should help but you should not exceed 0.05% sds and you should have 20% methanol in the transfer buffer, as well.

[Prep!]

mdfenko, on Mar 30 2010, 01:01 AM, said:

i agree with pi that sds should help but you should not exceed 0.05% sds and you should have 20% methanol in the transfer buffer, as well.

yes as a matter of fact we use 0.05% SDS with 20% methanol in the transfer buffer!!!