Fluorescent visualization of Western blot - (Nov/09/2009 )
Hi,
I have a suspicion for why none of our bands look strong using polyclonal antibodies, even those for so-called abundant proteins.
We have a good scanner (Fuji LAS-3000) that we use for densitometry of EtBr-labeled DNA gels, which has emission filters at both 520 and 580. So we use it to visualize bands on Western blot by using fluorescently labeled secondaries (Alexa 488). Should we expect this to inherently lead to less sharp bands, or even to less intense bands, compared to the intensity of background staining? (this could be the tradeoff in which fluorescent-labeling being an easier and less messy procedure than HRP)
Any information you can give about experience with fluor-conjugated antibodies versus HRP- or AlkPhos- conjugated antibodies, for visualizing protein blots, would be interesting. Thanks.
There is no reason why the signal should be worse using fluorescence c.f. luminescence, the antibody will bind the same, and the response should be similar. The signal will fade a bit with repeated exposure (or exposure to standard fluorescence lights), as it would in an IF experiment. The signal can be pretty weak, but it should be visible with the right excitation and emission filters. Your 520 filter should give plenty of signal, though the 580 will be a bit weak.
There are a few machines coming out now using IR wavelength excitation and fluorescent labels for westerns... they work pretty well from the demos I have seen.
MDavies on Nov 9 2009, 12:25 AM said:
I have a suspicion for why none of our bands look strong using polyclonal antibodies, even those for so-called abundant proteins.
We have a good scanner (Fuji LAS-3000) that we use for densitometry of EtBr-labeled DNA gels, which has emission filters at both 520 and 580. So we use it to visualize bands on Western blot by using fluorescently labeled secondaries (Alexa 488). Should we expect this to inherently lead to less sharp bands, or even to less intense bands, compared to the intensity of background staining? (this could be the tradeoff in which fluorescent-labeling being an easier and less messy procedure than HRP)
Any information you can give about experience with fluor-conjugated antibodies versus HRP- or AlkPhos- conjugated antibodies, for visualizing protein blots, would be interesting. Thanks.
Do you have the apropiate PVDF, most have autofluorescence.
Alexa 488 captures at 520?
Also you may need to try to let the scanner record for a long while and superimpose the images.
Still, ECL is usually more powerful than fluorescence. You should at least make a try with ECL to see that everything is ok but the fluorecence at that point.