Can somebody attach a step by step and thorough protocol for aqueous two phase partitioning of Arabidopsis root plasma membranes? I am not experienced and I need an easy to follow approach.
Hi George,
It has been a while since I last did Two-Phase partitioning and it is fairly late now, so I might make some stupid mistakes when trying to explain. The most important factor to take account of is the final concentration of polymers, which is species-dependent. As mentioned in Alexandersson et al. Plant Cell Physiol (2004) 45 (11): 1543-1556, the optimum concentration for A. thaliana is 6.1%:
"The resulting microsomal pellet was resuspended in 6 ml of resuspension medium: 330 mM sucrose, 5 mM K-phosphate, pH 7.8, 0.1 mM EDTA, 1 mM DTT (freshly added) and 50 µl protease inhibitor cocktail. The resuspended membranes (6.0 ml) were added to an 18.0 g phase mixture to produce a 24.0 g aqueous polymer two-phase system with a final composition of 6.1% (w/w) Dextran 500, 6.1% (w/w) PEG 3350, 5 mM K-phosphate, pH 7.8, and 3 mM KCl."
In practice you make stocks of 20% DT500, 40% PEG, 200 mM K-Phosphate pH 7.8 and 2 M KCl. With the aid of a pipette and a balance you make a mixture on a clear centrifuge tube adding from the stocks:
7.32g stock DT500 solution
3.66 g stock PEG solution
2.04 g solid sucrose
480 microliters K-Phosphate buff.
36 microliters of stock KCl
Distilled water up to 18g
This will be the first two-phase system (TPS) tube to be used. Shake the tube well and leave on ice until use.
To prepare the counter-current tubes, you mix (again on a balance):
14.64 g DT500 stock
7.32 g PEG stock
5.44 g solid sucrose
1280 microliters K-Phosphate buff.
96 microliter KCl
Distilled water up to 48g (edited on 19-XI-12)
Shake to mix and place on ice until phases separate. You may centrifuge (3000xg for 5 min) to help. Then, take the upper phase from the lower one and save them apart . Split the lower phase into two tubes identical to the one used for the first TPS tube (we will call them L2 and L3). The upper layer will be needed later too: they will make U2 and U3.
Prepare your microsomes and resuspend the pellet in 6 ml of 5 mM K-Phosphate pH 7.8, 330 mM sucrose. Add the well-resuspended membrane prep to the first TPS tube. Shake well and centrifuge (3000xg, 5 min).
Take the upper layer (U1) and place it on top of one of the two tubes containing only lower phase that you prepared earlier (i.e. L2). Shake well and centrifuge. At the same time, add half the volume of the upper layer you separated earlier and place it on top of the lower layer from the first TPS tube (this upper layer will be U2 and the lower layer will be L1). Shake and centrifuge.
Take U1 and place it on top of a fresh lower phase (L3); take U2 and place it on top of L2; place a fresh upper phase on top of L1 (i.e. U3). Shake them and centrifuge.
Take U1 from L3 and place it in a ultracentrifuge tube. Take U2 an place it on top of L3; take U3 and place on top of L2. Shake and centrifuge.
Take U2 from L3 and pool it with U1. Take U3 and place on top of L3. Shake and centriguge.
Finally take U3 from L3 and pool it with the previous two upper phases. Dilute at least three fold with a buffer of your choice (for exampl...
Hi George,
It has been a while since I last did Two-Phase partitioning and it is fairly late now, so I might make some stupid mistakes when trying to explain. The most important factor to take account of is the final concentration of polymers, which is species-dependent. As mentioned in Alexandersson et al. Plant Cell Physiol (2004) 45 (11): 1543-1556, the optimum concentration for A. thaliana is 6.1%:
"The resulting microsomal pellet was resuspended in 6 ml of resuspension medium: 330 mM sucrose, 5 mM K-phosphate, pH 7.8, 0.1 mM EDTA, 1 mM DTT (freshly added) and 50 µl protease inhibitor cocktail. The resuspended membranes (6.0 ml) were added to an 18.0 g phase mixture to produce a 24.0 g aqueous polymer two-phase system with a final composition of 6.1% (w/w) Dextran 500, 6.1% (w/w) PEG 3350, 5 mM K-phosphate, pH 7.8, and 3 mM KCl."
In practice you make stocks of 20% DT500, 40% PEG, 200 mM K-Phosphate pH 7.8 and 2 M KCl. With the aid of a pipette and a balance you make a mixture on a clear centrifuge tube adding from the stocks:
7.32g stock DT500 solution
3.66 g stock PEG solution
2.04 g solid sucrose
480 microliters K-Phosphate buff.
36 microliters of stock KCl
Distilled water up to 18g
This will be the first two-phase system (TPS) tube to be used. Shake the tube well and leave on ice until use.
To prepare the counter-current tubes, you mix (again on a balance):
14.64 g DT500 stock
7.32 g PEG stock
5.44 g solid sucrose
1280 microliters K-Phosphate buff.
96 microliter KCl
Distilled water up to 48g (edited on 19-XI-12)
Shake to mix and place on ice until phases separate. You may centrifuge (3000xg for 5 min) to help. Then, take the upper phase from the lower one and save them apart . Split the lower phase into two tubes identical to the one used for the first TPS tube (we will call them L2 and L3). The upper layer will be needed later too: they will make U2 and U3.
Prepare your microsomes and resuspend the pellet in 6 ml of 5 mM K-Phosphate pH 7.8, 330 mM sucrose. Add the well-resuspended membrane prep to the first TPS tube. Shake well and centrifuge (3000xg, 5 min).
Take the upper layer (U1) and place it on top of one of the two tubes containing only lower phase that you prepared earlier (i.e. L2). Shake well and centrifuge. At the same time, add half the volume of the upper layer you separated earlier and place it on top of the lower layer from the first TPS tube (this upper layer will be U2 and the lower layer will be L1). Shake and centrifuge.
Take U1 and place it on top of a fresh lower phase (L3); take U2 and place it on top of L2; place a fresh upper phase on top of L1 (i.e. U3). Shake them and centrifuge.
Take U1 from L3 and place it in a ultracentrifuge tube. Take U2 an place it on top of L3; take U3 and place on top of L2. Shake and centrifuge.
Take U2 from L3 and pool it with U1. Take U3 and place on top of L3. Shake and centriguge.
Finally take U3 from L3 and pool it with the previous two upper phases. Dilute at least three fold with a buffer of your choice (for exampl...
Hi Emilie,
Sorry, I forgot to add that detail (it was nearly 2:00 a.m. when I wrote the protocol and I was feeling quite sleepy).
Yes, the final weight of the stock for the counter current tubes is 48g. This way you end up with three tubes with similar concentrations and similar weights (24 g). The first TPS has apparently a mistake on the concentrations of phosphate and sucrose, but that is because your microsomes also contribute with these (6ml ~ 6g of a solution containing phosphate buffer and sucrose is added to the first TPS -16g- making it up to 24g).
I hope there are no more mistakes on my part, but please, if you find any, shout it!
Best wishes,
Agustín
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