Protocols for protein extraction from whole tissues for isoelectric focusing

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℃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 at37℃

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 2530℃ 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℃ to phenol phase.

7.Vortex and incubate at 20℃for at least 1 h or overnight.Collect the precipitate by centrifugation (20 min,20,000 g,4℃.

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℃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℃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 2530℃ 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.