THE UNIVERSITY OF CALIFORNIA AT RIVERSIDE

Standard Operating Procedure

K. Le Roch Laboratory

TITLE: PROTEIN EXTRACTION (after parasite extraction)

Page 1 of 1

SOP #: KLR5

REVISION LEVEL:

DATE:

1        MATERIALS

1.1       Lysis buffer

·      20 mM Tris 7.5

·      20 mM MgCl2

·      1 mM PMSF (or AESBF) + Protease inhibitor mixture...

·      10 mg/mL leupeptin

·      10 mg/mL aprotinin

·      10 mM NaF

·      15 mg/mL benzamidine

·      10 mM glycerophosphate

·      1000 mM ATP

1.2       Biorad dye reagent

1.3       SDS PAGE Gel

2        PROCEDURE

2.1       Resuspend parasite pellet in ~2 volumes of ice-cold lysis buffer

2.2       Homogenize by brief sonication @ 4°C (6 ´ 10-20 s, 20% sonication max, until the color turns grey/black)

2.3       Centrifuge 15 min @ 4°C and 20,000 g (max speed)

2.4       Supernatant is soluble fraction (cytoplasmic proteins), pellet is insoluble fraction (membrane proteins)