Rhabditid Vitellogenin Purification
(ref.: C.E. Winter - Comp. Biochem. Physiol., 103B:189, 1992)
OBS.: 1. This protocol should be followed when you have more than 3g of worms.
2. All steps must be carried at 4 oC to minimize protein degradation.
01. Pre-equilibrate a 10 ml ConA-Sepharose column with TH 1x.
02. After washing the worms over 30% sucrose, freeze them by droping the slurry
over liquid nitrogen. Store the small frozen drops at -70 oC.
03. Grind the frozen worms in a mortar and resuspend the powder (in the same volume
of the worm mass) with HOMOGENIZATION BUFFER 2x (HB 2x).
---------------------------------------------------------------------------
| OBS.: BEFORE ADDING HB 2x TO THE POWDER, ADD 100 mM PMSF, 1 mM pepstatin|
| and 1 mM E-64 to a final concentration of 1 mM, 20 uM and 20 uM in|
| HB 2x, respectively. |
---------------------------------------------------------------------------
04. Shear the DNA with a syringe to get rid of the viscosity.
05. Centrifuge the homogenate at 1,500 xg for 5 min at 4 oC.
06. Dilute the supernatant with HB 1x.
07. Centrifuge at 10,000 g for 5 min at 4 oC.
08. Using a peristaltic pump, apply the supernatant over the previously
equilibrated Con A SEPHAROSE column at a flux of 9.4 ml/h. OBS.: take
a sample (20 ul) of the homogenate for SDS-PAGE control.
09. Wash the column with 150 ml de HB 1X during 16h (overnight) at a slower flux.
10. Elute the vitellogenin bound to the column with HB 1X cotaining alpha-
methyl-manoside 0.5 M. Colect fractions of 2,5 ml (~15,6 min at a flux of 9.4 ml/h).
11. Take samples of 50 ul from each fraction for SDS-PAGE.Precipitate the samples
with 10% (w/v) TCA, wash with acetone, dry and resuspend in
50 ul of SDS-PAGE sample buffer. Run 15 ul of each of those samples in a SDS-PAGE.
COLUMN REGENERATION:
01. Wash with 200 ml of REGENERATION BUFFER 1 (RB 1) at 10 ml/h.
02. Wash with 200 ml of RB 2 at 10 ml/h.
03. Wash with 200 ml of RB 3 at 10 ml/h.
STOCK SOLUTIONS:
-----------------------------------------------------------------------------
pepstat 2mM E-64 2mM HB RB 1 RB 2 RB 3
(200 x) (200 x) (2 x) (1 x) (1 x) (1 x)
-----------------------------------------------------------------------------
pesptatin 2,8 mg - - - - -
E-64 - 1,4 mg - - - -
1M Tris pH 7,4 - - 10 ml - - -
Tris Base - - - - 2,4 g -
NaCl - - 17,5 g 5,84 g 5,84 g 11,7 g
1M CaCl2 - - 0,5 ml - - 200 ul
0,1M MnCl2 - - 5,0 ml - - 2,0 ml
HAc - - - 0,67 ml - 57,3 ul
NaOAc.3H2O - - - 1,13 g - 2,58 g
Boric Acid - - - - 1,2 g -
-----------------------------------------------------------------------------
Water to 2,0 ml 2,0 ml 250 ml 200 ml 200 ml 200 ml
-----------------------------------------------------------------------------
OBS.: 1. For eluting the bound vitellogenin:
HB 2x ....................... 50 ml
alpha-methyll manoside ...... 9.7 g
Water to 100 ml