Coupling BSA-Peptide #4 to CNBr-Sepharose

(Sat, March 11, 2000)

As a guideline, follow instructions in the Pharmacia booklet on Affinity Chromatography: principles and methods.

CNBr-Activated Sepharose = Sigma # C-5338

Volume Guidelines:

· Swell gel with 200 ml 1mM HCl per g dry gel, in multiple small washes, rather than a single large one.

· Perform coupling in 5 ml buffer per ml of swollen gel.

· For average protein ligands use 5 – 10 mg of protein / ml gel (roughly 1 – 10 umol protein). HOWEVER, if the column is to be used for Immuno-affinity purifications, then less ligand should be used. Also note that with peptides coupled to carrier proteins, the ligand is multivalent, since several peptides are generally coupled to a single carrier protein.

1. Swell CNBr-Sepharose:

· 0.25 g of gel in a 10 ml tube

· + 10 ml 1 mM HCl

· Incubate at 4 deg for 15 min on rocker

· Wash 5 x 10 ml 1 mM HCl

(To remove wash, spin 2 min. in clinical centrifuge at setting # 3 = approx. 700 g)

----prepare ligand in coupling buffer during this time----

· RECORD SETTLED GEL VOLUME = approx. 0.8 ml

2. Prepare Ligand:

Ligand = TLTF peptide #4, coupled to BSA via MBS.

Ligand concentration = 20 mg/ml

· 0.025 ml ligand (0.5 mg)

· add 1 ml coupling buffer ( 0.1 M Bicarbonate, pH 8.3, 0.5 M NaCl)

3. Rinse swollen gel with coupling buffer and add to ligand:

(***SINCE THE ACTIVE GROUPS ON THE GEL HYDROLIZE AT ALKALINE pH, THE PROTEIN LIGAND SHOULD BE ADDED AS QUICKLY AS POSSIBLE AFTER ADDING COUPLING BUFFER TO THE GEL.***)

· Following the final 1 mM HCl wash, rinse the gel with 5 ml coupling buffer

· Spin at setting #3 for 2 min.

· Resuspend in 2 ml coupling buffer.

· IMMEDIATELY Add 1 ml of ligand in coupling buffer

· Place on rocker at Room Temp for 2 – 4 hours.

TIME ON: 5:10 pm

TIME OFF: 7:55 pm

4. Block unoccupied active groups.

· Wash gel once with Blocking Buffer (0.2 M Glycine, 0.1 M Bicarbonate, 0.5 M NaCl, pH 8.0)

· Resuspend in 5 ml Blocking Buffer and rock at Room Temp for 2 hours.

TIME ON: 8:00 pm

TIME OFF: 10:00 pm

5. Wash away excess uncoupled ligand.

· Wash with 5 cycles of:

5 ml Coupling Buffer then 5 ml Wash Buffer (0.1 M Na-Acetate, 0.5 M NaCl, pH 4.0)

· Wash 5 x with 5 ml Coupling Buffer.

· Wash 2 x with sterile PBS + 0.025 % NaN₃, pH 7.3.

· Final bed volume = _____0.5 ml____

· Store at 4 deg in PBS, azide.