Acetone protein extraction

[rohit14]

Hi Guys,

I need some assistance.....

Protocol that I am referring to says to “add 3 volume of acetone to the supernatant of homogenate and incubate at 4C for overnight to precipitate proteins”. I am talking of larger volume around 10 liters. Now, my questions are:

1. Can we do this in shorter time period by mixing slowly at -20C for 30 min to 120 min or by increasing acetone to 4 volumes? There are some general
protocols that states that above mentioned is doable but I am not confident.
2. Will it interfere with the lipid removal as protocol itself does not refer anything about the lipid removal and I am not confident if by changing the volume or time/temp, may ppt lipid also and then it will add to impurities in protein ppt.

Please help with your experience and any scientific literature with web-link.

Thanking you all in anticipation.

[mdfenko]

until i knew more about why things are done a certain way, i would stick with the tried and true protocol. sorry.

[MT Scientist]

Sounds like a lot of acetone. Perhaps it would be useful to do an intermediate purification step such as "salting out" (i.e ammonium sulfate precipitation)....

[relaxin]

I have done this when I was a graduate student. Yes, it is a large volume of acetone. You need to use acetone that has been stored at -20 C. But you need not wait overnight for protein to precipitate. I tbink 30 min should be long enough. Then we collect the precipitate by filtering through a huge filter funnel lined with filter paper using a suction flask and vacuum. When all liquid passed through, rinse the precipitate with more cold acetone, and then air dry. The product is a white powder. If you use acetone at room temp, the whole thing will turn into a brown gluey mess.

[rohit14]

Thanks Everyone. I know this is quite a volume for low flash point chemical.

Thanks very much Relaxin for your suggestion. I have 4 questions for you if you can please answer them:
1. Were you using 3 volumes or 4 volumes of acetone to precipitate the protein during 30 min incubation?
2. Were you stiring the extract for 30 min or left it at stationary condition?
3. I am going to dissolve it in Phosphate Buffer (0.2M/pH 7.0). Do you remember what buffer you used to dissolve the white ppt?
4. Did you had to add acid/base to dissolve this white ppt and how readily it was able to dissolve?

[relaxin]

1 and 2. I check back my dissertation, I actually used 5 volume of -20 C acetone. There was no mention of incubation time. I did the precipitation in batches using a 4-liter plastic beaker. I just stirred well and recovered the precipitate with Whatman #1 filter paper in a Buchner funnel. I started one beak of acetone precipitation while I filtered the previous one. So the stirring time is 30 min at the most. When all precipitate are collected, I rinsed it with cold acetone and allowed it to dry in vacuuo (with continuous suction).

3. The choice of buffer used to dissolve the precipitate depends on the solubility of your protein. I used 0.2 M ammounium acetate, pH 6.8 and ran gel filtration with the same buffer. Not all the precipitate will be dissolved in the buffer. You need to adjust the pH of your suspension and centrifuge to remove the insoluble material prior to running on columns. Otherwise proteins start to precipitate when the pH of the protein solution changes as it moves down the column. Keep the insoluble material frozen until you are sure you got the protein you want. You can do another extraction with buffer of a different pH, if necessary.

4. My protein is very soluble at pH 6.8. However, since it was extracted using HCl-acetone, I need to add NaOH to adjust the pH to 6.8. As I have said, not all precipitate is soluble, so I just centrifuge to remove the insoluble material (it is a purification step!).

[rohit14]

Thanks very much Relaxin, thanks a lot....

This was a great help. I would give it a try.... -20C/30 min/4 volumes.

Last question for everyone: Can we centrifuge a large volume of acidified Acetone protein extract?

Thanks Guys !!

[mdfenko]

yes

[MT Scientist]

Last question for everyone: Can we centrifuge a large volume of acidified Acetone protein extract?

The question would be: Do you have a big enough centrifuge and proper bottles to centifuge the extract?

[relaxin]

If your centrifuge can only hold 250 ml centrifuge bottles, you can still do it batchwise (200 ml per bottle, 1.2 liter per run). Overfilling the centrifuge bottles may cause leakage. You need to centrifuge at 4 C, because the heat generated will denature the protein and create problem with acetone vapour.

[rohit14]

Hi Guys,

MT Scientist- Yes, I have 4 refrigerated centrifuge with 1 liter x 6 rotor and so I can easily do 24 liters refrigerated centirfugation at a time. So that is not a problem as it can be set at 4200rpm/5100g at -15C but acetone being so volatile (more than ethanol) is my concern.

Thanks again Relaxin and MT Scientist and other members. I will update you guys once I have some success.

[ikin]

My company also used very large amount of acetone. They try to recycle it from the waste
, did you guys have any ideas?