eIF4G western blot - (Dec/13/2011 )
I am having problems running eIF4G. Where I normally have a perfect line, I am seeing a bleached out area with a "blob" looking line underneath my target area. I have tried a different antibody from another company and still get the same results. This is happening to all my samples for this specific initiation factor. Any suggestions would be helpful. I have attached a picture of what I normally get and what my blots look like now. Thanks!
The bleached area is probably from the blot not being covered properly by the primary antibody solution. You may have to try titrating the antibody to remove some of the background you are seeing.
the blank areas below the bands look like bubbles. the membrane may not have been in full contact with the gel during transfer.
Thanks for the responses! I don't think its contact or bubbles. I have run 20 or more of these and I get the same results. I tried adding a NaCl solution and it removed the blob but still nothing in my target area. I've ran other factors and those seem to come out great. I have run the corresponding 4E factor on a WB with the same samples and that comes out fine too.