negative band on film

[alexr]

Hi,

i have a problem...
i use a HRP-coppeled WB system und it does work right goog.
on this film here i got a very high background and negative band. i mean the band are white on a black background.

i do use TBS...

anybody any idea?

thanks

alex

[kmunson779]

Try using less luminescent substrate and/or shorter exposure times.

[kevin_a]

Actually the substrate is not the cause of the problem here.

You have negative bands because there is a slight background developing do to some nonspecific binding of the HRP on the membrane. You have less signal where your bands should be creating the "ghost bands".
If you would like to see some examples try this url www piercenet com/files/AN0011dh5.pdf

Reason - the enzyme has used up all of the substrate in the area where the bands should be OR the enzyme has been oxidized because there is too much enzyme in the band location.

Another possibility issue is that the HRP conjugate is not labeled well and the little amount of HRP present is getting inactivated quickly.


The solution to this issue is to use less HRP conjugate. During your Western, dilute the secondary more. If you are using 1/5,000, try 1/10,000 (1/10,000 then try 1/20,000). You might have to try several before you get it optimal.

Sometimes you can rinse the blot off with PBS and then soak in substate again to see some signal. This is not going to be optimal because of the ezyme oxidation issue. If you see yellow or brownish colored areas on the blot, you can be sure you've oxidized the HRP, but this is not always apparent.