Baculo purification

11/15/04

 

  1. After 72 hours infection, spin down cells in 2 50mL tubes and the rest (3L) in 500mL bottles.

 

  1. 50mL conical add 10mL lysis buffer. Dounce x20

 

  1. Sonicate 3x30 seconds and rotate for 20Õ at 4 degrees.

 

  1. Spin 12Kxg 30Õ 4 degrees.

 

  1. Preequilibrate beads in lysis buffer. Spin 2K, 3Õ.

 

  1. Incubate supe with 400uL 50% slurrry glutathione beads for 1 hour at 4 degrees.

 

  1. Take supe and repeat incubation with 400uL beads for 1 hour. (Repeat x5). For each binding save 50uL beads and 50uL supe as diagnostic.

 

  1. Pool beads, wash 4X with lysis buffer (500mM NaCl total). 10mLs per wash.

 

  1. Elute on column with 1 mL elution buffer x 4.

 

  1. Protein assay elution fractions and run on SDS-PAGE gel versus BSA standards (0.1-2ug).

 

  1. Stain coomassie, destain and view.


 

 

 

Lysis Buffer (50mL)

2.5mL 1M Tris pH8.0

1.5mL 5M NaCl

250uL 1M DTT

5mL 7% CHAPS

5mL 10% Tx-100

1 protease inhibitor tablet

250uL PMSF

ddH2O to 50mL

 

Elution buffer:

100mM Tris 8.0

20mM GSH