protein extraction - (Jun/30/2008 )

Hi to you all,

I have some old samples of organisms that are preserved in:
1. 24 hours in 40% formaldehyde and sugar
2. Ethanol 70% + 1% glycerin
In room temperature.
Do you think I can still extract proteins from these animals? I would like to do "western" with those samples in order to estimate HSP, do you think it is doable with old samples?

Thanks in advance

---

I am not sure but I can see two problems:

1. Proteins crosslinked with each other, so tehre may not be separation of proteins necessary for western.

2. Epitope recognition would go haywire, due to crosslinking.

No harm in trying though. If your samples are valuable, just treat a sham sample in a similar manner and see if it works for the proteins of your interest with then antibodies you have.

---

as cellcounter implies, FA promotes cross-linking of proteins, and may cause problems; I fear as there seems to be little prevention of oxidation damage, you will not have much fun with your extraction

---

thank you both very much, i will try with similar treated sample and see.

---

If you are planning a sham experiment, you can do the following too while you are at it.

1. Run on a low percentage gel. It is more likely than not that the protein of your interest is crosslinked with a bunch and may not even move from the well.

2. Transfer for a longer time in that eventuality.

3. If separation is not as important to you as detection, also do a slot blot.

4. In ChIP, we treat chromatin with high-salt, 65'C >4 hours for formaldehyde de-crosslinking, perhaps you can treat a tube in a similar manner to see if your protein gets at least partially de-crosslinked and runs on the gel.

---