Coupling Protein to Cnbr Activated Sepharose Beads

Coupling protein to CNbr activated Sepharose Beads

Dissolve 5mg of peptide in 0.5-1.5ml of 0.1M sodium phosphate buffer pH 6.5-7.5. Follow procedure given with peptide. Dissolve the peptide gradually.

*if won’t dissolve, add one drop of HCl to decrease pH. Once dissolved, must bring pH of buffer back up to 6.5-7.5 by adding drops of NaOH*

*after dissolved, do a dot test on nitrocellulose and/or 4ul for a protein assay to check how much peptide dissolved*

Procedure:

Stage 1:

1) Weigh out 0.3g of dry CNBr activated Sepharose (use a 2ml Eppindorf tube if volume of dissolved peptide is less and a 15ml Falcon tube if volume is larger.) (0.3 g of resin can bind approximately 5mg of peptide)

2) Wash beads with 2ml of 1mM HCl at 6000rpm for about 5 seconds. Repeat 10 times. Remove HCl.

3) Add 0.5ml of 0.1M sodium phosphate buffer pH 6.5-7.5. Immediately spin down the resin and remove the supernatant and add the dissolved peptide.

4) Place on a rotating platform in the cold room for 6 hours or overnight.

Stage 2:

1) Spin down the resin at 6000rpm for 1 minute.

*save a sample of the supernatant to assay for uncoupled protein via dot test and/or protein assay*

2) Add 0.4ml of 1M Monoethanolamine (NH2(CH2)2OH) pH 7.5. (blocks any reactive sites that have not bound proteins or hydrolyzed water.)

3) Place on a rotating platform in the cold room for 6 hours or overnight.

Stage 3:

1) Spin down the resin at 6000rpm for 1minute and remove the supernatant.

2) Wash the 3 times with 1xTBS.

3) Keep the resin in 1xTBS in the fridge until needed.