PhosphoaminoacidanalysisofnonTCAprecipitableproteins

Phosphoamino acid analysis of non-TCA precipitable proteins

1. Cut out gel slice, incubate 60 min with 1 ml 5.7 M HCl @ 110oC in 13 x 100 mm screw cap tubes.

2. Add 4 ml water, centrifuge @ 2000 rpm in bench top centrifuge for 10 min, transfer supernatant, freeze and lyophilise.

3. Dissolve residue in 10 ml water, add 0.4 ml 50% (v/v) slurry of Dowex AG 1 X 8 (Cl- form), adjust pH to 7.5 - 8.5 with NH4OH.

4. Add 1 μl of solution containing 1 mg/ml pSer, pThr and pTyr. Shake at room temperature for >4 hr.

5. Settle resin, wash into a blue tip + frit (6 mm), wash twice with 0.5 ml water, count tip.

6. Elute phosphoamino acids with 2 x 0.5 ml 0.1 M HCl.

7. Count eluate and lyophilise.

8. Resuspend in 6 μl pH 1.9. buffer, spot 4 μl after spinning.

Alkali treatment of gels

Add 500 ml 1 M KOH to gel, place in 55oC oven for 2 hr. Pour off KOH, and replace with normal destain for 2 hr with changes until gel is back to original size. Rinse with water and dry