Can i recover protein from a stained SDS-PAGE? - (Aug/28/2007 )

hello all,

I have a band on SDS-PAGE that i would ideally like to recover and run again on a gel. The original gel has already been stained with colloidal coomassie, and destained with 25% methanol. I have heard of protein diffusion and electroelution, but have very limited knowledge of these techniques.

Could someone please advise to whether this is possible, and if so point me in the direction of some protocols that may be useful? I have tried to google these methods, but jsut get company websites coming up etc.

thanks in advance for any help,

L

---

i would say no because for coomassis coloration, usually there is a solution (acetic acide +++) which makes the protein precipitates and so not mobile.
I'm not sure, but in MS, the protein is digested in gel before recovery of peptides out of the gel. You may ask MS specialist to see if it's possible to recover your protein.

---

Thank you, i did wonder about the coomassie staining. you are right, the protein was first fixed in 7% TCA in 40% methanol, stained, and destained in 25% methanol.

what exactly does the TCA do?

---

normally the protein precipitates in situ in presence of acid which prevents protein diffusion

---

I'm very sorry, but i don't quite get it. where does it go when it precipitates?

---

well after you run your gel, acid slowely enter in it during stain. When reaching proteins, their conformation change (same way as if they were in solution i guess). It's irreversible as i know, and it's called "in gel precipitation"

---

However, if you're planning on doing more work, it's presumably on the denatured protein, because it's denatured by the SDS-PAGE.
You can crush PAGE slices, and recover from there, but it's often not a very high-yielding process.

---

hello,

thank you for your answers. I think i have decided to try this protocol anyway;

* place excised gel piece in microfuge tube

* Add 0.5 -1ml elution buffer (50mM tris-hcl, 150mM NaCl, 0.1mM EDTA; pH 7.5) so that gel piece is covered.

* crush gel pieces with pestle and incubate on rotary shaker overnight at 30'C

* centrifuge at 5000-10000 g fot 10 min and pipette supernatant into new tube.

Now, my questions are, what is the easiest way to make up the elution buffer? 0.1mM EDTA is a tiny amount, and i probably need 1ml of the buffer at the most!

also, is the salt going to be a problem when i re run this on SDS-PAGE?

Thanks again!
xx

---