Western blotting background troubleshooting - (Jun/22/2004 )

to reduce the background you can may be try Bloc ace instead of milk or BSA . I tried it and it workd well.
Goood luck

Hi Raje,

I feel your blocking step is too short and thats the reason you get cover.Keep the membrane in blocking solution for long time, preferably over night in 4 degree.Wash steps are important again to remove unbound antibody . Hope it will work.Use 5 % non fat milk powder and 0.1% Tween 20. Cheers!!!

Saikat

Mie University

Japan

I use 5% milk in TBST, yust as you did, but I incubate 1 hour! It works for me.
The first thing I would try is a different dilution of the antibody (less antibody), second I woud raise the amount of BSA or milk you incubate your antibody in (BSA protects against aspecific binding).
Third: Wash longer on the last wash; that removes a lot of background.

Good Luck!

Great topic for discussion. In my experience, the secondary antibody is generally the cause of high background, given ample washes. I no longer use Santa Cruz, Upstate, Cell Signaling, or Abcam secondaries but do use Jackson Laboratories secondaries. Link http://www.jacksonimmuno.com/home.asp

I use their secondaries at 1:10,000 dilution and get no background.

One other thing I do is to rinse membranes in di H20 2-3 times after wash in TBST.

Good luck!!

Once I tried secondary 1:1000, and everyhting turns black. It obviously too high, try at least 1:2000. Milk or other blocking must be in any ABs solution. We usualy use both primary and secondary in 1% milk (in PBST), and 1% is usually enough, but for blocking still use 5% milk.

I know someone do western without transforming protein to membrane. Is there someone konw this IN GEL western and how to do that?

If you are having dirty blots you may want to give the blots a good wash after the primary antibody, maybe 3 10 min washes? also in your protocol you seem to interchange the use of TBST and PBST is there a reason why? normally people use TBST if they are working with phospho antibodies

Where is your secondary antibody from? And which species? Also, where does your dry milk come from?

Thank you so much. Smaragdas.

Look at my topic Western Blotting: Sharing experience.