Dominik Haudenschild August 11, 1993 at CBRC

1) Weigh out amount of Sepharose. 1g = 3.5 ml

-1 to 10 mmoles (prefer 2) per ml of gel

-equivalent to 5 to 10 mg protein per ml of gel

2) Swell gel for 15 minutes in 1mM HCl. 20ml per gram

3) Wash gel with 1mM HCl. 200ml per gram

4) Dissolve protein in Coupling Buffer

-gel:bufer volume ratio should be 1:2

5) Immediately before adding protein solution, wash gel with

5 ml coupling buffer per gram dry gel

6) Without delay add protein solution to gel.

7) Incubate on end-over-end rotator 2 hr RT or overnight 4'

8) Hydrolyze residual active groups in Tris-HCl pH 8.0 at RT 3 hr

OR Block groups in1M Ethanolamine pH 8.0 at RT 3 hr

9) Wash with five cycles of coupling buffer then Acetate Buffer

10) Store at 4' in neutral pH, avoid Sodium Azide as bactericidal

agent as it will compete off the coupled protein.

NaCl 0.5M = 7.3g

pH 8.3 adjust with NaOH

Acetate 0.1M = 1.44ml Glacial Acetic Acid

NaCl 0.5M = 7.3g

pH 4.0 adjust with NaOH

HCl 1mM = 20.8 ml of Conc. HCl

Glycine 0.2M = 0.75g

pH to 8.0 with NaOH

Dominik Haudenschild August 10, 1993

Use 10 column volumes TBS pH8.0

If really dirty do 2 cycles of coupling then Acetate Buffer

Rinse again in TBS pH 8.0

Add solution containing protein to be purified. pH should be

around 8 for good antibody interactions. NaCl concentration should be 0.5M for minimum non-specific

interaction with column.

Wash column with TBS (pH 8.0 ; 0.5M NaCl) until wash does

not foam when shaken (indicating little protein)

Elute with 8M Urea, 50mM Tris pH 7.5, collecting 0.5 to 1.0ml

fractions. Alternative, stronger Elution buffer is 1M Acetic Acid.

Clear column with TBS, then add Anti-Bacterial Agent, (Not