Hi,
I am doing a pulse chase experiment and looking for 35-S methionine/cysteine incorporation in my protein of interest. Following gel run, I directly dry the gel (10% SDS-PAGE, 1.5mm thick, BioRad mini) in a gel drier for 1 hr at 80C. Then I expose the Xray film with an intensifier screen. My signal intensity is either very weak or no signal at all even after longer exposure (I went up to 72 hrs.). Although, several things might impact the result, however, I was wondering if fixation is necessary after the gel run (before gel drying, I guess)? If so, how would you do that?
I would appreciate any comment/suggestion!
Thanks
Sumit
Hi Sumit Mukherjee . If the gel is dried the proteins are not going anywhere. You could try incubating the gel with scintillation fluid before drying.
I agree that fixing is not necessary.
Film can be made more sensitive in 3 ways
1 intensifying screens as you did
2 Pre flash the film will increase sensitivity,
3
Put the cassette in a -80 freezer
Steps 2 and 3 should increase the sensitivity by a factor of 4-8
I think it would be a good idea to fix the gel before drying it in order to prevent diffusion of the bands before the gel is fully dried. Gels can be fixed by putting them in destain (50% methanol, 10% acetic acid by volume in water) for a short time (e.g. 30 min). Then, the gel should be soaked with water to remove the methanol and acetic acid before drying.
- I agree with Adam B Shapiro that fixing is a good idea, especially for low MW proteins.
- Also, perform a conventional staining of a gel, to make sure you actually have bands.
- An intensifying screen will not harm, but probably give little benefit. The ceramics in them produces light when struck by high-energy radiation like those produced by 32P. The interaction cross-section of such rays with X-ray film is relatively low.
- Autofluorography is a must for very soft radiation like 3H, which is absorbed by the gel and never reaches the photographic film. It may also be beneficial with medium hard radiation from 35S. 33P or 14C. You can use PPO (doi:10.1111/j.1432-1033.1974.tb03599.x), but also the water-soluble salicylic acid (doi:10.1016/0003-2697(79)90716-4), which doesn't require the use of DMSO.
- Incubation at -80 °C in combination with very long exposure times (2 weeks or more) can increase sensitivity.
- Buy pre-flashed film, this is more reliable than flashing it yourself. Pre-flashing sensitises the grains of AgBr, fewer photons are required to produce a silver grain. The trick with flashing is to use enough photons to increase sensitivity, but not so many that background is increased.
- Electronic detectors (phosphorimagers) are more sensitive than X-ray film.
Fixing is an integral part of the procedure. Adam has explained why. Please read this document. You just might find what you need. http://www.ispybio.com/search/protocols/CPMB.Ch.App3.Techniques.pdf.
Hello, plastic foils or glass plates is necessary for fixation of polyacrylamide gels to the support autoradiography. When using glass plates as support, autoradiography creates problems which can be completely avoided when the autoradiography plates are heated for 30–45 min at 55–60°C prior to gel polymerization.
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