||Weigh accurately sufficient quantity of the sample containing above 10mg protein in a thick-walled heavy glass tube having a constriction at 7.0cm from top.
||Add 5mL of 6N HCl and place the tube in a liquid nitrogen.
||Evacuate the frozen sample to 0.01mm Hg.
||Thaw the sample by shaking the slight warning to allow any dissolved air bubble out.
||Freeze the contents and evacuate to 0.005mm Hg again.
||Seal the tube at the constriction using a flame till the tube is under evacuation.
||Place the tube in a hot air oven at 110 ±1°C for 22h to hydrolyze protein
||Break open the seal and evaporate the contents in a rotary evaporator to remove hydrochloric acid.
||Add water and repeat evaporation two times.
||Take the residue in 1-10mL 0.01N HCl or 50mm citrate buffer (pH 2.2)