how to recover my protein? - A terrible mistake (Jan/12/2007 )

Hello,
I´ve just make a mistake. I was adding Bradford reagent to quantitate protein concentration, when, by mistake I added Bradford also to my original samples. How to recover the protein? I´m trying to precipitate it with TCA (overnight). Do you think will it work? Or it will be useless...

Thanks a lot

---

what are you thinking about?

---

the good news: bradford is a non-destructive method for protein determination.

the bad news: it is very difficult to remove coomassie blue (cbb) (g-250) from proteins.

the tca should precipitate the protein but it should still be dyed. what are you planning to use it for afterwards?

if it is to be used for sds-page then it should be no problem (the cbb should migrate ahead of the sample and bromphenol blue). native page methods, however, will not work properly.

tca precipitation will denature the protein (if it hasn't already been denatured by the methanol and phosphoric acid of the bradford reagent), so it shouldn't be viable for enzyme assay (unless it is a substrate, then it may be okay).

will it work? it all depends on what you need the protein for.

---

Thank you, mdfenko, for the information,

I was going to use the protein sample to isoelectrofocuse (IEF) (1D), just before being electrophoresed in a SDS-PAGE gel (2D).
I don´t know up to what extent the coomassie dye affects the net charge of the proteins, in any case I guess the migration shouldn´t be affected too much, and the proteins would be focused in a proper way. What do you think about it?

Thank you for your time

---

i think the tca may ruin your protein for ief. if you can renature your protein from tca (not impossible) then it may work, most of the cbb should release from the protein during electrophoresis.

you can try but start thinking in terms of getting fresh protein.

---

Any idea to remove Coomassie dye from the proteins?

---

i've used an electrophoretic concentrator to remove the dye and concentrate the protein (we would electroelute stained protein from acrylamide gels, concentrate and remove dye in one step). but the dye should release when you run the protein on a gel.

---

So, according to your answer, it would be possible to recover the protein by renaturing? Do you know how to get it?. At the end, I need to do the IEF only to asses some conditions of protein processing previous to the focusing, that´s to say, it isn´t a definitive experiment.

Thinking of getting fresh protein is depressing (several days and high-cost of money)
Sniff, sniff

---

you might be able to renature your protein. if you tca precipitate then you will first have to remove the residual tca (acetone extraction, i think, might also be ether extraction). then find a way to resolubilize the pellet (may have to denature then renature, urea then dialysis to refold properly). the dye may be gone by this time, or not.

on the other hand, you may be able to dialyze your protein in buffer to remove the methanol, phosphoric acid and unbound cbb. then load the protein on the ief gel.

i don't know if any of this will work but you can try.

but, i repeat, you may want to get started on a new isolation.

---

Finally, I decided to start again with fresh proteins.

Anyway, I apprecciate your help a lot

Thank you

---