Injecting [35S]methionine to measure neurofilaments- How? - (Mar/20/2010 )

Hi!

I am studying for my pharmacology test. It's an article of an experiment where they try to measure the levels of NF-H (Neurofilament-Heavy) protein in neuronal cells.

The thing is, they say that they do this by injecting <35S>methionine and then measuring the radioactivity.

Obviously, this is a radioactive version of methionine.

However, I can not understand how injecting this in cells can help measure NFs. What's the connection? The paper doesn't say, and I couldn't find the answer around the web.

Thanks

-seriousperson-

OK after reading a section again, I understood that by injecting this radio-methionin they label NF-H so that they can then detect this protein.

The paper sais:

Proteins <...> were metabolically labeled and fractionated <...>. Aliquots of 0.5 mCi of <35S>methionine <...> were injected <...>. 30 d after labeling, the sciatic nerves <...> were dissected out <...> cut into <...> consecutive segments. Proteins were fractionated by SDS-PAGE and radiolabeled proteins detected by fluorography.

(cutted out some unrelated sections to reduce copyright problem>.

Now my question is, how do they know that this isotope will target NF-H and not other proteins in the neurons?

-seriousperson-

After many hours and much searching around, I managed to put together a large part of the answer.

They injected radiolebelled methionine in mice. Methione is an amino acid that gets can get incorporated into a protein during this protein's translation. It can thus be used to detect proteins. So they then isolated the nerves of interest. They used SDS-PAGE to fractionate the proteins in gel. They used fluorography on the gel (where the proteins where already separated and could be identified due to their mass) and they thus detected all proteins of the neurons.

I also have the question of how exactly does an amino acid get "incorporated into a protein during its translation".

If anyone knows the answer to this please let me know. For now I'll continue looking :lol:

EDIT: I found that using tagged amino acids to incorporate into target protein is called "metabolic labeling".
But still haven't found how this works.

-seriousperson-

Of course, I'm stupid, the answer is simple:

tRNA takes the labelled amino acid and it gives it to the ribosome when the corresponding codon is read. The ribosome thus inserts it in the polypeptide chain.

So I guess I answered my question myself, after several hours.

It was nice talking to my self here. Goodbye.

-seriousperson-