Antibody purification

Exchange the buffer of the MBP fusion protein using pD10 columns

• Equilibration buffer:

100 mM HEPES pH 8.0 (shelf!) 500 mM NaCl (2.922g NaCl for 100 ml; M=58.44g/mol) 10 mM Maltose (M=360.32g/mol; shelf: 20g/100ml⇒ 20%) Add CaCl2 to 80 mM final conc. Save an aliquot for later analysis ⇒ Add in a later stage!

• Buffer exchange:

equilibrate PD10 column with 4 CV (column volumes) 2.5 ml of protein solution (if less, fill it up to 2.5ml with buffer) Elute protein with 3.5 ml of equilibration buffer Add CaCl2 to 80 mM final conc. to protein solution

• Cleaning of PD10 columns:

Wash with minimum 4 CV 1x PBS Wash with minimum 3 CV 10x PBS Store the columns in 10x PBS at 4 C

Affinity purification of antiserum on antigen coupled NHS HiTrap columns (Drechsel, A. Desai)

Solutions (sterile filtered):

- 1mM HCL (prepare fresh; shelf:1M)

- 0.1 M M glycine pH 2.6

- 0.1 M triethylamine pH 11.5 (δ=0.728g/l)

 (13.89 ml for 1l of solution)

- 2 M Tris pH 7.0 (or 100µl Tris pH 9.0 + 900µl Glycin, check pH! ) - 10 mM Tris pH 8.0

- 10x PBS

- 2x PBS

- 20% NaN3

- Blocking buffer 0.5 M ethanolamine (3.06 ml 100% EtOHamine for 100ml solution) 0.5 M NaCl

- Wash buffer 1x PBS 0.5 M NaCl 0.1% Triton X 100 (=10 ml Triton X-100 pure)

- Storage buffer 1x PBS (from 10x stock) 55% glycerol (v:v)