I'm blotting against STAT3 and pSTAT3Y710. My pSTAT3 antibody works great, but I observed a negative band (a white band that is lower intensity than background) at the predicted MW for STAT3 when blotting for total STAT3. What could be the cause of this, and more importantly, how do I fix it?
are you doing SDS PAGE ? May be the first thing to do is look and see if there is protein in that band - there are several protein stains for membranes - try one and let me know what you get
Agree with Paul. Try a simple Ponceau S with the membrane, or even Coomasie S in the gel. If there is a protein load in the range, you have something which binds to that protein and blocks the detection Ab.
Hi Carl,
I have sometimes observed this if the signal is to strong. The films are 'burned' in this area. This can usually be seen by eye, the bands show a green fluoresence if you look at them in the dark.
Are you using old school films? If so lower the exposure time (down to putting it on for only a second). If this gives you a black band reduce primary and/or secondary antibody or switch to a less sensitive ECL.
Hello
I agree with researchers may you have a too much protein or as a suggested too much antibody
Good Luck
Hello,
I think you should try to visualize it with a digital device that will enable you to play with the exposure time and background.
Good luck
Hi Carl,
high amounts of antigen concentrated in a single band will bind high amounts of enzyme-linked antibodies that rapidly uses up the substrate thus causing a "negative" band at this position of the blot. Wash the blot, use fresh substrate and expose for a very short time period. Or repeat the blot using 10x lower amounts of antigen.
Best luck, Ingrid
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