Can someone provide me some information about detection of bands in WB by fluorescence? I'm trying to resolve my issue that I don't have any other means of quantitation.
Hi Ngyen;
Not sure what you are asking exactly. If you want to visualize your total protein profile on a Western blot, then SYPRO Ruby blot stain will do that. A simple set-up involves a UV transilluminator and a Wratton #9 filter placed betwen your imaging device and the blot. Alternatively, a blue light transilluminator, like the Dark Reader (Clare Chemical) is a fairly economic set-up for this application.
Fluorescence imaging and quantitation of Western blots is available with some commercial gel documentation systems, although these are quite expensive instruments. Here is one supplier:
https://www.azurebiosystems.com/imaging-systems/
The Cary Eclipse is really for solution phase fluorescence assays, not for blots. I suppose you could attempt to elute the fluorescent dye off of individual bands and quantify, however this is really the wrong instrumentation for Western Blot analysis. You’d be better off using a colored stain, like Amido Black, and a cheap flatbed document scanner.
Depending on what membrane do you have. If you use the PVDF, after developed, the membrane can be kept in the air and dried.
ECL (enhanced chemiluminescence)-developed blots can be imaged using an expensive cooled CCD camera system (excellent for quantitation), or exposed to X-ray film. The developed x-ray film can be scanned with a flatbed scanner for quantitation and for recording the image digitally. Film is not a great medium for quantitation because it has a small dynamic range, but it is a lot cheaper than a cooled CCD camera.
Expose the blot for several different amounts of time with different sheets of film (or different parts of the same piece of film if it is a small blot) to be sure of getting a good exposure before the luminescence fades. The ECL reagent instructions will suggest a suitable range of exposure times, which depends on the reagent. It may be anywhere from a few seconds to a few minutes. Some reagents continue to luminesce for a long time, others only for a few minutes.
Don't let the ECL reagent come into direct contact with the film. The blot has to be wrapped in clear plastic before being brought into contact with the film.
Your last resort is to develop the blot with a chromogenic reagent. That requires no equipment for visualization, but it has poor quantifiability. It is not as sensitive as ECL.
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/chromogenic-western-blotting-substrates.html
You also need the capability to develop the film. You can use the old-fashioned manual method with trays full of chemicals, but there are also automated developers. You also need a dark room. It can be illuminated with a red lamp, since x-ray film is not sensitive to red light, but white light must be completely eliminated.
Yes. You just need to select a suitable fluorogenic enzyme substrate. If the plate reader is a filter-based instrument, make sure you select a substrate for which you have the right filter set.
I have another problem, could you let me know if I can use this product
Goat Anti-Rabbit IgG (H + L)-HRP Conjugate #1706515
http://www.bio-rad.com/en-us/sku/1706515-goat-anti-rabbit-igg-h-l-hrp-conjugate?ID=1706515
to detect the primary antibody? In a website, it says , Secondary antibodies are specific for the isotype (class) and the species of the primary antibody (for example, a goat anti-rabbit secondary antibody is an antibody generated in goat for detection of a primary antibody generated in a rabbit)
In order to detect a plant protein, what secondary antibody should I use?
Thank you so much. I really appreciate your help. I'm looking at different options and thinking I should be back to Western blot.
The source of the antigen is not relevant. If you are using a polyclonal antiserum raised in a rabbit, the goat anti-rabbit conjugate should work well, since the antibodies are almost always IgG. The reagent for fluorescence detection must be one that is intended for use with HRP (horseradish peroxidase).
I have another problem, My primary antibody is this product http://www.enzolifesciences.com/ADI-SPA-818/hsc70-plant-monoclonal-antibody-1d9/Here is the information about this product
HSC70 (plant) monoclonal antibody (1D9)
Host:Mouse Isotype:IgG1 Immunogen:Recombinant spinach Hsc70 (Hsp73). Species reactivity:Porcine, Plant Applications:IP, WB Recommended Dilutions/Conditions:Western Blot (1:1,000, colorimetric)
I will buy this secondary antibody:
Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP: blotting-grade horseradish peroxidase secondary antibody conjugate
http://www.bio-rad.com/en-us/sku/1706516-goat-anti-mouse-igg-h-l-hrp-conjugate?ID=1706516
Will my secondary antibody react with my primary antibody? I am talking about doing colorimetric and then scanning. Thank you very much. Thank you so much.
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