Protocols for protein extraction from whole tissues for isoelectric focusing
Proteomics Center
12-12-02
SDS extraction followed by acetone precipitation – simple extraction protocol that does
not require phenol. Recommended start protocol for whole tissue extractions.
1. Grind 1 g of fresh tissue to a powder with liquid nitrogen in a mortar and pestle.
2. Add 5 mL of extraction media (0.175 M Tris-HCl, pH 8.8, 5% SDS, 15%
glycerol, 0.3 M DTT) directly to mortar and continue grinding for an additional
30 sec.
3. Filter homogenate through two layers of miracloth into a 50 mL Falcon tube at
room temperature.
4. Immediately add 4 volumes of ice cold 100% acetone to filtered homogenate, mix
by vortexing and place at -20 C for at least one hour to precipitate proteins.
5. Centrifuge at 5000 g for 15 min to collect precipitated protein, decant supernatant.
6. Gently blot residual acetone from container with Kimwipe and then wash pellet in
15-20 mL of cold 80% acetone. Be sure to thoroughly break-up pellet by
pipetting, vortexing or sonication.
7. Repeat steps 5 and 6.
8. Collect final protein precipitate by centrifugation at 5000 g for 15 min and dry
pellet by inverting on Kimwipe for 15 min at room temperature.
9. Resuspend final pellet in 0.5-1 mL of IEF extraction solution (8 M urea, 2 M
thiourea, 2% CHAPS, 2% Triton X-100, 50 mM DTT, 0.5% pH 3-10 ampholytes)
by pipetting and vortexing at 25-30 C. Incubate sample for 1 h at room
temperature with agitation. Do not heat sample under any circumstances as
this will lead to carbamylation of proteins.
10. Centrifuge for 10 min at 12000 g and use supernatant to rehydrate IPG strips.
11. If protein quantitation is necessary, precipitate protein sample with TCA or
acetone prior to performing Bradford or Lowry assay as detergents and reducing
agents interfere with these assays. Alternatively, use the new protein quantitation
kits (e.g. EZQ from Invitrogen) that tolerate detergents and reducing agents.
Phenol extraction followed by methanolic ammonium acetate precipitation – an effective
protocol for sample preparation from protein-poor, recalcitrant tissues such as plants (see
Hurkman and Tanaka, 1986, Plant Physiology 81:802-806).
1. Grind 1 g of fresh tissue to a powder with liquid nitrogen in a mortar and pestle.
2. Add 2.5 mL of Tris pH8.8 buffered phenol and 2.5 mL of extraction media (0.1
M Tris-HCl pH 8.8, 10 mM EDTA, 0.4% 2-mercaptoethanol, 0.9 M sucrose) and
continue grinding for an additional 30 sec in a fume hood. Alternatively, transfer
to a 15 or 50 mL Falcon tube and homogenize in polytron homogenizer for one
minute. For liquid samples, dilute 1:5 with extraction media and add equal
volume of phenol.
3. Transfer to Falcon tube and agitate for 30 min at 4ºC.
4. Centrifuge 10 min at 5000 g, 4ºC.
5. Remove phenol phase (should be top phase) and back-extract aqueous phase
with 2.5 mL + 2.5 mL of extraction media and phenol by vortexing. Centrifuge
and combine with first extraction.
6. Precipitate phenol extracted proteins by adding 5 volumes of 0.1 M ammonium
acetate in 100% methanol (stored at –20 C) to phenol phase.
7. Vortex and incubate at –20 C for at least 1 h or overnight. Collect the precipitate
by centrifugation (20 min, 20,000 g, 4 C).
8. Wash the pellet 2X with 0.1 M ammonium acetate in methanol, 2X with ice-cold
80% acetone and finally 1X with cold 70% ethanol*. Completely resuspend the
pellet each time with vortexing and if necessary, sonication (this usually takes
longer the first time). Place the resuspended sample at –20 C for at least 15 min
between each wash. A final wash in 80% acetone/10mM DTT can improve
protein solubility in IEF buffer. You can store the last resuspended pellet in the
acetone -80 C until ready for IEF.
9. Centrifuge to recover precipitated protein, remove acetone and dry the pellet
under nitrogen (or on the bench for 15 min). Do not dry too long (dry time will
vary based on the size of the pellet).
10. Resuspend final pellet in 0.5-1 mL of IEF extraction solution (8 M urea, 2 M
thiourea, 2% CHAPS, 2% Triton X-100, 50 mM DTT, 2mM tributylphosphine,
0.5% pH 3-10 ampholytes) by pipetting and vortexing at 25-30 C. Incubate
sample for 1 h at room temperature with agitation. Do not heat sample under
any circumstances as this will lead to carbamylation of proteins.
11. If protein quantitation is necessary, precipitate protein sample with TCA or
acetone prior to performing Bradford or Lowry assay as detergents and reducing
agents interfere with these assays. Alternatively, use the new protein
quantitation kits (e.g. EZQ from Invitrogen) that tolerate detergents and reducing
agents.
Notes:
 Preparation of samples must be performed with labware that has never been in
contact with nonfat milk, BSA, or any other protein blocking agent to prevent
carryover contamination.
 Always use non-latex gloves when handling samples, keratin and latex proteins
are potential sources of contamination.
 Never re-use any solutions, abundant proteins will partially leach out and
contaminate subsequent samples.