Can't detect GAPDH (western) with Alexa488 secondary antibody - (Oct/21/2014 )

I ran 10ug total protein (from RIPA harvest) my lanes and used Kaleidoscope protein standard.  I can detect the fluorescence within the Kaleidoscope standard at the low molecular weights, and the colors from the standard are very clear on the PVDF membrane, suggesting a good transfer.  However, I am unable to detect GAPDH using a anti-GAPDH (Cell Signaling) antibody @ 1:1000 and Invitrogens Alexa488 secondary @ 1:2000.    I know fluorscence is inferior to chemiluminscence in terms of amplifying a signal, but 10ug protein should be sufficient to see a band.  

When I place my western on a blue light transilluminator I can clearly see the fluorescence from the standard; however, there is no signal in the body of the blot.  I attempted to detect the bands using an EpiBlue filter on a gel imaging system using 100ms exposure.  Is this long enough?

100 ms might be too short an exposure, can you try longer ones, or some sort of integrative imaging (ones where each image that is taken is added to the last)?

bob1 on Wed Oct 22 20:08:26 2014 said:

100 ms might be too short an exposure, can you try longer ones, or some sort of integrative imaging (ones where each image that is taken is added to the last)?

I had my blot sitting in TBST overnight and reimaged this morning using a 1 second exposure and there is nothing.  I'm very suprised that I cannot detect GAPDH in 10ug protein

Assuming your antibodies are working, you should be able to see it quite clearly. Using an Odyssey system (IR based fluorescence) I would do a 2-5 min exposure for most proteins. I've never actually used any of the visible light based ones for this sort of thing.

bob1 on Wed Oct 22 22:40:57 2014 said:

Assuming your antibodies are working, you should be able to see it quite clearly. Using an Odyssey system (IR based fluorescence) I would do a 2-5 min exposure for most proteins. I've never actually used any of the visible light based ones for this sort of thing.

One thing I did in an attempt to save time was mixed my primary and secondary antibody together and incubated the blot at 4C for 2hrs.  I read on a BiteSizeBio thread that this is acceptable since the primary recognizes the antigen and the secondary recognizes the Fc domain.  I've done hundreds of Westerns in the past (the current one is the first I've done in several years) but this is something I've never tried before.  I'm inclined to think that this may have messed something up, but practically I cannot think of a reason why.  I have some lysate left and I will retry, except I will do the primary and secondary separately and I will use an HRP secondary.  

I would have thought that 2 hours might be a bit short at 4. You could try at room temp. I have done the primary/secondary mix thing before and it has worked for actin, but I haven't tried it with other antibodies.

are you sure the secondary only recognizes the Fc? if it was made against the whole molecule then it may be neutralizing the primary.

also, the primary-secondary complex may be coming out of solution. you may want to use a little more vigorous shaking and/or more incubation time (overnight?).