Optimizing AmAc concentration for protein precipitation
Date: 07/30/20
Testing material: Clarified MT302 lysate in 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors) (1:4 powder to buffer ratio; sonication 5x 2sec, @ 2Amp; Spin @ 20k rcf for 10’ @ 4°C)
Take 4 phasemaker tubes, add 25ul water and then 50ul Chloroformand 250ul of Trizol
Mix by hand, vigorously for 15 sec
Spin 16k RCF, 4°C (not important, Trizol extract is stable at RT), 5 min
Add 200ul of cold ammonium acetate in methanol (AmAc/MeOH) with different concentration (0.1, 0.2, 0.5, 1M)
Mix by hand, vigorously for 15 sec
Spin 16k RCF, 4°C., 5 min
The organic phase moved to the top. Transfer the top layer to a fresh tube
pH of the mix was 6, between 6-6.5, 6.5 and 7 respectively with increasing AmAc concentration
Add another 625ul of AmAc/MeOH of the same concentration to each tube Also add 0.5ul of MT302 lysate and 1ul of glycoblue in each tube; Leave the tubes in -20°C freezer overnight for protein precipitation
Check the pH, choose AmAc lowest concentration of that can best neutralize the organic phase (pH 7) pH of the mix was 6.5, 6.5 to 7, 7 and 7 respectively with increasing AmAc concentration
Precipitate was pelleted, 20k x g for 30' @ 4°C.
Make the side of Eppendorf tube that the pellet should be at; carefully remove the supernatant
After centrifugation, wash pellet 1x with 0.1M AmAc/MeOH, spin 15’ @ 4°C
Wash pellet 1x with Acetone, spin 15’ @ 4°C
Dry the pellet under vacuum
Resuspend the pellet in 10ul of LDS and analyze samples on gel with MT302 lysate (load 0.5, 0.2 and 0.1ul equivalent) to check precipitation efficiency
Take 1ul of MT302 lysate, add 8ul of 1.1x LDS and 1ul DTT, take 5ul, 2ul and 1ul and add more 1.1x LDS to each tube to 10ul
Add 1ul of 0.5M DTT to all samples
Heat samples @ 70°C for 10’
Run 15-wll 4-12% Bis-Tris gel with Acquastain
1) Marker
2) MT302_0.5ul_0.1M AmAc
3) MT302_0.5ul_0.2M AmAc
4) MT302_0.5ul_0.5M AmAc
5) MT302_0.5ul_1M AmAc
6) Space
7) MT302_0.1ul
8) MT302_0.2ul
9) MT302_0.5ul
10) Space
11) BSA_50ng
12) BSA_100ng
13) BSA_150ng
Notes and discussion:
We can draw the following conclusion from the experiment above:
(1) the pH does not make that much of a difference 6.5-7 is fine
(2) adding more AmAc, even if it helps pH be closer to 7 also does not improve things
(3) In the context of this experiment it didn’t matter when the glycoblue was added - but I would simply say that, in general, it makes sense to add it after recovery from phase maker, before adding extra alcohol.
This is not a well-planed experiment.
The glycoblue should be added AFTER the phase maker but BEFORE adding the additional 4 vols of MeOH
I was just checking the final pH by adding AmAc/MeOH with different concentration. When I notice the difference in pH, I thought that maybe I can check the precipitation efficiency as well. So, I added 0.5ul of lysate and 1ul of glycoblue here. The right way of doing the experiment should be:
1. Add 0.5ul of lysate to 250ul of Trizol, mix well
2. Add 25ul of H2O, 50ul of ChCl3 to phasemaker
3. Add 250ul of Trizol with sample (step 1) to phasemaker, mix well
4. Spin and separate the aqueous phase and organic phase
5. Remove aqueous phase; Add 1 vol. of AmAc/MeOH to organic phase
6. Spin and move the organic phase to top
7. Transfer the organic phase to a fresh tube
8. Add glucoblue and additional AmAc/MeOH; Mix well
9. Incubate in -20C overnight
About the amount of AmAc/MeOH added
There has been some variability in the results up to here… so, I still feel a little anxious but the 5 vols with 0.1M and glycoblue seems to be the best.
Current amount of AmAc/MeOH added was based on the calculation: Everything we added to the phasemaker gave a total volume of 325ul (25ul H2), 50ul CHCl3, 250ul Trizol). The aqueous phase we recovered is around 160ul (similar volume based on Lars' record). So I assumed the the organic phase was ~165ul since I could not get an accurate measurement by passing the pipet tip under the phasemaker gel. Actually, after adding 200ul AmAc/MeOH to the organic phase and making it move to the top. I usually get 310ul back (organic phase + 200ul of AmAc/MeOH).
I tried mixing 25ul H2O, 50ul CHCl3 and 250ul Trizol and separating directly by centrifugation without phase maker. The organic phase was ~130ul. Since we were adding 825ul of AmAc/MeOH to the organic phase, we added 6.35 volume of AmAc/MeOH to precipitate protein.