Protein Preps
Drop Dialysis
Drop dialysis is a fast way to remove salts or other small molecules from small volume samples. The sample, from 20 to 400 ul, is placed on top of a filter that is floated on buffer in a Petri dish.
1. Add 20-30 ml of 0.5X TE to a 100 mm Petri dish. Place the Petri dish where it will be undisturbed by contact or vibration.
2. Using blunt forceps, gently place
a filter (Millipore cat # VSWP 02500) on top of the solution with the shiny
side facing up. If more than one sample will be dialyzed simultaneously, the
filter should be marked with pencil or
water proof pen prior to application of the sample. Allow the filter approximately
one minute to fully wet.
3. Slowly pipet the sample onto the surface of the filter, using a wide-bore pipet tip. Cover the dish and allow to dialyze from 20 minutes to two hours at room temperature.
4. Remove samples with a wide-bore pipet tip, expect to recover about 90% of the sample volume. Do not try to remove the entire sample, since this is likely to submerge the filter with resulting loss of all remaining samples.
Notes:
Up to four samples of 50 ul each can be placed on a single filter. Brief dialysis,
less than 1 hour, may increase the sample volume if the applied sample contains
a high concentration of salt or sucrose. Prolonged dialysis (more than 4 hours)
will result in loss of sample volume due to gravity.
Protein Precipitation Methods
When using TCA wear lab coat, glasses and work in a fume hood. When working with TCA keep a supply of sodium bicarbonate nearby in case of spills.
TCA-DOC (low volume protein
concentration)
1. To one volume of dilute protein add 1/100 vol 2% DOC in ddH2O (sodium deoxycholate).
2. Vortex and let sit for 30 min on ice or at 4 C.
3. Add 1/10 volume 100% TCA and incubate overnight at 4 C.
4. Centrifuge at max speed for 15 min at 4 C.
5. Pour off the supernatant and dry pelley with a Kimwipe.
6. Wash the pellet twice with with starting volume of -20 C acetone.Incubate
the pellet for 15 min on ice, and centrifuge for 5 min max speed at 4C for each
acetone wash.
7. Kimwipe the samples and and dry the pellets in a fume hood.
If your protein is soluble in acetone, resuspend the pellet in 100 mM Tris pH 8.0. Wash the pellet with diethyl ether. Then add 2x loading buffer and run proteins on a gel.
To look at samples on a gel - TCA
method
1. Make protein solution 13% with TCA
2. Incubat for 5 min at –20C and 15 min at 4 C
3. Centrifuge at max speed for 15 min at 4 C
4. Wash and dry with acetone as above.
Acetone Precipitation
1. To 1 volume of protein solution, add 4 volumes of cold acetone.
2. Incubate overnight at -20 C.
3. Centrifuge and wash once with acetone as above.
Large volume method
1. In a 30 ml corex tube, add 6.4 ml of ice cold methanol to to 1.6 ml of sample
and vortex mixture.
2. Add 1.6 ml ice-cold chloroform and vortex.
3. Add 4.8 ml ice-cold DI H2O and vortex.
4. Incubate on ice for at least 5 min.
5. Centrifuge at 9000 x g for 5 min.
6. Discard the upper phase (the protein is at the interface, do not discard).
7. Add 4.8 ml of ice-cold MeOH.
8. Centrifugre at 9000 x g for 10 min.
9. Carefully pour off the MeOH.
10. Dry the pellet.
For 100 ul of sample use 400 ul of MeOH, 100ul of chl and 300 ul of H2O.