Increase Exposure, Bands Disappear??

[Mediatorx]

I have been running lots of western lately with no problems....until now.
It is time to re-run blots to get "the pretty picture", so I am using the same samples, membranes, primary, secondary, buffers, detection reagent, etc. When I develop, I usually exposure for 30 seconds to make sure things are straight, then 3 minutes, then 10 minutes (my protein is not so abundant). Generally, I will only see my standard (MagicMark) at 30s, faint bands at 3min, and pretty nice bands at 10min. Now, I barely see the standard at 30s, at 3min I see the standard OK, and see my bands faintly, but at 10 minutes I see nothing. It isn’t background blown out--it is just all white--you can see the outline of the membrane if you crank up the gamma. But, no bands are visible, no sample bands and no standards. I originally thought that our imager must be broken, but no one else in the lab is having this problem. I have even borrowed their blots and exposed them repeatedly, and their bands only get darker with increased exposure time. What is going on with my sample?? Any ideas on why the bands would completely disappear between a 3min exposure and a 10min one?

[canuck1043]

I'm not 100% sure what method you're using, but I've had this with ECL and tubulin...apparently if there is too much protein, (and subsequently antibody) the luminescent enzyme gets 'eaten' up during the reaction and runs out in a very brief period...

[fatima]

or your ECL is bad. Get some if you can from other lab and try it

[kevin_a]

This problem could be caused a few things.

If I remeber correctly, MagicMark is made of fusion proteins with Protein A or G so that the bands bind your secondary antibody.

If the substrate is bad, the signal can fade quickly.
If there is too much HRP, the signal can fade quickly.
If the HRP conjugate is poorly labeled, the signal can fade quickly.

An easy check of the substrate is to put 1ml of mixed substrate in an 1.5ml tube, go into the dark room and add 1ml of HRP-conjugate. If the signal is weak and short, try again (fresh substrate) with 1 or more additional HRP conjugates. If all these test are short, the substrate is lost its juice. If the signal is bright and steady for a minute or so, the substrate is good.

Sometimes you can see signal again after washing the membrane with PBS and then reincubate in fresh substrate, but this is not ideal as some of the enzyme will have lost activity due to oxidation (HRP substrates).

Assuming the substrate is good, dilute out your secondary antibody out the next time.
If that doesn't help, you might want to try a different HRP conjugate. Some are poorly conjugated and the signal you see in the test tube test is not always an indication of conjugation efficiency since most companies do not remove the Free HRP from the antibody conjugate.