Protein extraction from whole tissues for IEF

 

Modified from that of Jay Thelen - University of Missouri-Columbia

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).

Grind 1 g of fresh tissue to a powder with liquid nitrogen in a mortar and pestle.

Add 2.5 ml of Tissue Extraction Medium and continue grinding for an additional minute in a fume hood. 

Transfer to a 15 ml Falcon tube and add 2.5 ml of Tris pH8.8 buffered phenol and mix.

Sonicate for 30 s at setting 60 and then agitate for 30 min at 4 C.

Centrifuge 10 min at 6000 rpm (SS-34), 4 C.

Remove 1 ml of the phenol phase (should be top phase) and place in a new 15 ml Falcon tube. 

Precipitate phenol extracted proteins by adding 5 volumes of ammonium acetate in 100% methanol.

Vortex and incubate at -20 C for at least 1 h or overnight.  Collect the precipitate by centrifugation (10 min at 6000 rpm (SS-34), 4 C).

Resuspend each pellet in 600 ul of ammonium acetate in methanol and transfer each to a screw-cap microfuge tube.  Completely resuspend the pellet using a P200 set at 150 ul.  Add an additional 600 ul of ammonium acetate in methanol.  If the pellet is not resuspended completely, either sonicate briefly (at 40) or place in the sonicating water bath for 5 minutes. 

Place at -20 for at least 15 minutes and then collect the precipitate by centrifugation  for 5 minutes at 12,000 rpm in a microfuge at 4C.

Repeat this washing process a second time with ammonium acetate in methanol, 2X with cold 80% acetone, and finally 1X with cold 70% ethanol. 

Dry the final pellet briefly at 37 C after removing as much of the ethanol as possible with a Pipetman. 

Resuspend final pellet in an appropriate (minimum) volume of IEF Sample Buffer and store at -20 C. 

protein extraction solutions

1 M Tris-HCL (pH 8.8) - 78.8g/500 mls

Tissue Extraction Medium (100 ml) - filter sterilize and store at 4 C

0.1 M Trizma base pH 8.8 1.21 g
10 mM EDTA   2 mls of 0.5 M stock
0.9 M sucrose   30.8 g
0.4% B-mercaptoethanol add 40 ul to 10 ml of buffer just prior to use

prepare Tissue Extraction Medium without the mercaptoethanol which should be added just prior to use

you need 2.5 ml of this buffer for 1 g of tissue

Tris- saturated phenol (pH 8.8) - store at 4 C

take a bottle of phenol and put a stir bar in it
add an equal volume of 100 mM Tris pH 8.8
stir overnight
repeat
remove most of the Tris leaving a cm layer to cover the phenol

your need 2.5 ml of phenol for 1 g of tissue

0.1 M ammonium acetate in 100% methanol (100 ml) - store at -20 C
0.77 g/100 ml of methanol

80% acetone (100 ml) - store at -20 C

70% ethanol (100 ml) - store at -20 C

IEF Sample Buffer (10 ml)

8 M urea  4.85 g
2 M thiourea  1.52 g
2% CHAPS  0.2 g
2% Triton X-100 200 ul
50 mM DTT  8 mg/ml
make to 10 ml with ddH2O
make this buffer without the ampholytes or DTT and store at room temperature
add 8 mg DTT/ml just before use
add 0.5% ampholytes just before use 5 ul/ml (3-10 or 4-7 depending upon strip)