Hi everyone,
we're planning to do some protein quantification using western blot. Since a lot of our proteins of interest are of similar size, I would either have to strip and reblot a lot or run a number of gels on samples with quite limited amount of protein.
I've worked with ECL so far, but discovered that our imager is actually capable of fluorescent western blotting. Some of the primary antibodies are of different species (mouse and rabbit) and from the reading I've done, I could use this to multiplex my approach with two differently fluorescent secondary antibodies, right?
I would like to combine these two secondary antibodies with a measure of total protein quantification, as there are quite a few products available (TGX Stain Free Gels from BioRad, No Stain from Thermofisher, REVERT from Li-Cor), I was wondering if someone versed in the field could give me suggestions on what to look for.
Our imager is capable of Blue, Green, Red, NIR and UV light.
I was planning of using a Alexa Fluor Plus 680nm and Dylight PEG 800nm secondary antibody.
Thank you!
I have done some fluorescent WB, they work fine but you need to take into account several things :
1. Your membrane : nitrocellulose membranes are fluorescent and interfere with the analysis, PVDF is slighly better but not optimal. There are membranes that have less fluorescence (GE, fisher etc., slighly more expensive).
2. Try to use freshly made buffers, clean tubes and so, try not to touch your membrane with bare hands, and your migration dye front is fluorescent too, sometimes i let it run out of the gel in order to get clear results.
3. Your proteins can be of similar size, but not too close, otherwise your fluorescence signals will interfere and superpose, but you can change reading parameters (voltage etc.)
4. Regarding the antibodies, what you say is correct, but you 'll need to try and see if these antibodies are really specific or not, Alexa Fluors have worked quite nice. And your choice looks OK, try to use antibodies fluorescent in the red spectrum (in the blue and/or green, some proteins are intrinsically fluorescent (ex.mitochondrial ones etc., in the red spectrum you are not bothered by that).
5. Since you mentioned fluorescence quantification, bear in mind that the signal may not be linear, so you may need some work with that
Otherwise, for the stain-free quantification, several products have worked nicely for me, so up to you to choose what you like.
Thanks for the detailed reply!
1). Yes, I tried conventional PVDF membranes and found them to have quite a bit of autofluorescence in the red channel (I've ordered low fluorescent alternatives).
3). I will run a few trials on cell lines, to see whether signal interferance between the different proteins is a problem.
4). Thank you, I got the impression that a lot of people were using 680 and 800nm, which seemed a bit weird to me coming from flow cytometry, but this makes sense.
5). Could you suggest a product that you have found to work (and that would work with my antibody selection)? I was thinking about buying BioRad TGX Stain-Free gels, they aren't too open about what wavelength to expect, but supposedly UV light / Blue light should work. With Thermos alternative I'm concerned that 590nm is too close to my red secondary.
For Li-Cor REVERSE it is implied in the name, that you can wash it off after taking the image, but I haven't found any mentioning, that you can actually do that and not run into problems with the 680nm secondary.
Thanks a lot!
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