Hi, I am going to use the extraction procedure from an article, however, I don't understand the bolded step. Could you please explain how I precipitate the protein in cold acetone and what the 5:1 ratio means? Thank you so much.
Frozen tissue was ground in buffer 1.5 mM TRIS-HCl, pH 7.5, 2 %w/v) Triton X-100 and 0.1 % v/v) 2-mercapthoethanol), in a ratio 1 : 2 w/ v). The mixture was then heated at 100 C for 90 s and centrifuged at 15 000 g for 5 min. The supernatant was transferred to a fresh tube and the protein was precipitated with cold acetone in the proportion 5 : 1 acetone : supernatant. The protein was resuspended in 100 mM TRIS buffer pH 6.8)
Hi there,
5:1 is the ratio between acetone and sample volumes. If sample volume is 1mL then acetone volume is 5mL. Incubate the mixture on ice and collect precipitate by centrifugation. Dry the pellet before reuspension is the desired buffer.
Dear Nguyen,
In addition to the above. Just for clarity:
In the first mentioned buffer your protein of interest is soluble and present after centrifugation (in the supernatant). This so-called supernatant (that contains your protein) is put in another tube and apparently becomes insoluble in aceton (called precipitation).
As said above suppose your supernatant is 1 mL you have to use five times more (cold) aceton (so in this case 5 mL). The text don't mention it but you have to perform a (slight) centrifugation step in order to get a pellet with your protein that is then again made soluble in the next mentioned buffer.
Hope it is clear now what is the basic thought behind this.
Good luck!
You collect the precipitate by, as I mentioned, an extra centrifugation step (just try and see whenever your solution (the supernatant) becomes clear).
Resuspend means that you bring the protein (that is in a pellet in your tube after centrifugation) back in solution. With the buffer as mentioned in the article (the 100 mM Tris etc.).
Hope it is all clear to you now.
Yep ;-)
That's why Dominique already mentioned that you have to dry the pellet (to get rid off the residual aceton etc.).
Good luck!
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