getting rid of nonspecific background in westerns - (Jul/20/2007 )
I have problems with westerns at the moment. to get rid of background and other non-specific bands I was thinking of pre-incubating the Primary Ab diluted in milk with whole cell extract from a strain deleted for the protein I am probing. Has anyone tried this? and do you have a clue as to how much protein I should be adding to my dilutions?
I have also tried blocking with 7% skimmed milk in TBS-T but my westerns after developing came out blank any suggestions?
I have done almost precisely what you are thinking of with excellent results! I wouldn't recommend a direct incubation of the antibody and lysate. Rather, I ran tons and tons of cell lysate which didn't have my desired protein on gels, transfer and blotted the membrane with the antibody. It took a few gels/membranes before all the background went away but eventually all I had with the real experiment was one, perfect, gorgeous band! I've also seen this happen by reusing the working dilution many times. I'm not sure how much antibody you will need to add to the dilution to get a good working solution..that's going to take some trial and error. Start at a low dilution and keep adding until you see good results. I'm also not sure what's going on with the blank westerns... did you ponceau and see good transfer? Can you get any signal from the membrane? (when I get a blank I blot for actin just to see if the membrane is good) Some antibodies prefer PBS-T or maybe the antibody isn't good for westerns. Do you have another you can try? Did you run enough lysate? Maybe your ECL just isn't sensitive enough to get a signal. Unfortunantly there are many, many, many reasons why you can have a blank western.
For you normal westerns, you could try diluting the primary antibody further. Also you could block with 4% BSA.
Good Luck !!!
Hi Thanks for the suggestion.... i tried out the direct incubation and got beautiful bands on a test run i hope to get good results for my actual experiment! how long do you think the antibody is fine till you start see this background issue? Thanks