Protein isolation

[roth317]

Hi,

I am trying to quantify HIF 1 alpha protein level. Our scaffold is not a polystyrene plate, and the cells are not grown as 2D monolayers (I am looking at cell aggregates to be specific). Our original plan is to isolate the protein using trizol extraction reagent, then run an ELISA on HIF 1 alpha. However, I am worried that the protocol may not work. I haven't heard of trizol being used for a specific protein extraction, and HIF 1 alpha seems to be favorably isolated with western blot. If anyone has any input on this, I would greatly appreciate it. Thank you.

[JackBean]

How can you isolate any protein with Western blot?

Trizol should work for protein extraction, but I have never used it. I guess they will be inactive if is it OK for you.

[roth317]

Okay, after doing some reading, I have come up with a different plan for quantifying HIF-1 alpha, which involves running an ELISA after performing a nuclear extraction. However, all the protocols and kits I have seen recommend 1-10 million cells for a good extraction. I am likely going to be using much fewer cells than this number (as few as 50,000 cells). I am wondering if there are protocols or kits for nuclear extraction and ELISA that can be used with fewer cells. Any help would be appreciated.