we labeled a protein by iodine 125 for a goal and after labeling we want to see the protein band on SDS-PAGE again but after usual staining and electroforesis there is no band, however the iodine radiation indicate the existence of protein.
radio labelling is tremendously sensitive especially if you expose the film for days so an amount that gives a good signal may simply be well below the level of protein that can be stained chemically. What amount of protein were you labelling and how much did you run on the gel that you are trying to stain?
Protein staining methods differ in sensitivity. Coomassie staining is less sensitive than silver staining. There are also fluorescent stains that are of similar sensitivity as silver stain. Western blotting using an antibody specific to your protein can be extremely sensitive.
If you would like to determine the molecular weight of your labeled protein, run it on a gel along with molecular weight markers. Stain the gel with Coomassie Brilliant Blue to visualized the markers and then submit the gel to autoradiography to visualize your labelled protein. overlay the film with the stained gel to add the position of the standards onto the film. Of course, to do thins, you need film, and exposure cassette, and a developer. Alternatively, if you can find someone who has a phosphor imager, you can acquire the 125-I signal using a phosphor screen - without the need for film etc.
Bruce
one small detail to add to Bruce's answer. It makes orientation easier if you dilute a little labelled protein in blue dye and prick the gel with a needle wetted in it in 3 unimportant places then you get hot spots on the film to align with blue spots on the gel
Another small detail: you can avoid having to Coomassie stain the gel in order to see the marker protein ladder by using pre-stained markers.
Dears all Thanks a lot
Maybe i didn't explain the problem correctly. I don't have any problem with my protein to stain, all errors occur after labeling the protein with iodine125. I want to see after labeling if i have the appropriate concentration of protein per ml or not. Routinely i use 1 mg/ml of protein to label with iodine125 by chloramine T method and finally i use 10 micro-lit of labeled protein to run on the SDS-PAGE.
Don't you think that Chloramine-T interfere the staining process?
what volume of protein are you labelling please?
You may simply not have enough labelled protein to see using routine chemical staining
if you want to measure the amount of protein could you use uv spectrometry. It is more accurate than gel staining for quantification but in answer to the question I would not expect chloramine T to have any effect but I have only labelled ng quantities of protein in ul volumes and do not have any experience in using such large volumes for labelling
chloramine T can oxidize your protein; did you stop with an agent like sodium metabisulfite? did you run the labeling reaction over a desalting (PD10 or similar) column? if so, I wouldn't expect much if any chloramine T in the final product, so it shouldn't be the problem.
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