Western blotting background troubleshooting - (Jun/22/2004 )
I would like to remind a lazyman's solution to Western background. There are always steps to optimize - reagent amount, wash, incubation, exposure, etc., but don't forget a simple way to solve the problem - electronically edit your blot image. You can see the difference in the attached photo.
this is cheating!!
this is cheating!!
and many journals won't accept a heavily doctored figure.
No, the level function of any graphic program will reduce the background noise and enhance the signal. Why? Because noise is universal in the image but signal is uique. By electronical editing, which is the same as we include controls in biology studies, you are not cheating but getting to the truth in the your data. Just ask FBI 
I was joking. But more to the point, if you edit an image you must edit the entire image and not just part of it. The only tools I use on Photoshop would be the level function or the contrast, and only very conservatively at that. Imagequant has a function for elimating background and this is just fine to use because its applied to the whole image. But you have to be careful. I would not, for instance, use the color picker tool in Photoshop to chose a particular shade of gray and then elimate all areas that have that color because in that case you are only altering part of the image rather than the whole. The image that you provided looks like you did just that. Sorry to say, but it just looks too good.
I had the similar problem with high background (blot is dark in colour). It happened after I stripped the blots.
I washed it thoroughly to about 20 minutes per wash (3 times).
Is it possible if I have too long incubation hour for my primary or secondary antibodies?
is it because of the stripping part?
Any inputs are much appreciated.
I washed it thoroughly to about 20 minutes per wash (3 times).
Is it possible if I have too long incubation hour for my primary or secondary antibodies?
is it because of the stripping part?
Any inputs are much appreciated.
you may have stripped the blocking. did you reblock after stripping?
I only blocked it once. Is it necessary to reblock again? Hmm it makes sense now. I will try to reblock it. thanks
hie friend..i'm also hv problem w western blot.the background was bad and wht should i do..can u suggest and wht u'r opinian from this picture..
pls find the attachment..pls help meeee
tq u so much
pls find the attachment..pls help meeee
tq u so much
it looks to me as though something (either antibody or, more likely, substrate) precipitated on the membrane.
can you give information about your procedure (buffers, washes, antibodies, substrate, with shaking, temperature, etc.).