Protocol Online logo
Top : Forum Archives: : Immunology and Histology

ELISA wash buffer alternates - (Jun/13/2006 )

Regular reader, occasional poster...
I've been having a few problems of high background in an ELISA assay I'm re-working. I think I have weak interactions from "normal" samples. Does anyone have any suggestions? I'm considering changing the wash conditions such as 0.05% to 0.1% Tween in PBS. Any other buffers I should try?

Go Australia!


hi swanny! instead of changing your buffer increase your T-20 conc. to 0.1% or increase your washing time. background is usually the result of
1.poor blocking
2. improper washing
3.too much of primary antibody
4. or too much of secondary antibody
later two can be takebn care of with proper washings or increasing the conc. of your detergent.
you may also increase your blocking agent conc. this will not help much if you are already using optimum (usually vary from 2- 5%) concentration and still you are getting high back this case the culprit is definitely improper washing.
click here. this topic has already been discussed earlier.
how ever tris buffer saline with T-20 is another buffer of choice to perform ELISA


Thanks very much.