Affinity purification: Difference between revisions
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#*Dilute ligand (eg. FAA-N-GST) 1:10 in pH 10 Coupling Buffer. Reconcentrate to 1ml in 50mL 10Kmwco Centricon | #*Dilute ligand (eg. FAA-N-GST) 1:10 in pH 10 Coupling Buffer. Reconcentrate to 1ml in 50mL 10Kmwco Centricon | ||
#*Make 1:3mL dilution of exchanged sample. This is the Ligand Solution. | #*Make 1:3mL dilution of exchanged sample. This is the Ligand Solution. | ||
#'''make slurry''' | #'''make slurry''' | ||
#*Put 2-5mL of Coupling Gel into a 14mL conical centrifuge tube. | #*Put 2-5mL of Coupling Gel into a 14mL conical centrifuge tube. | ||
#*Wash w/ 5mL Coupling Buffer pH 10. Centrifuge for 5-10minutes at 1000xg. Aspirate supernate carefully. | #*Wash w/ 5mL Coupling Buffer pH 10. Centrifuge for 5-10minutes at 1000xg. Aspirate supernate carefully. | ||
#*Add Ligand Solution. Rock for 4hours at 4∞C. | #*Add Ligand Solution. Rock for 4hours at 4∞C. | ||
#'''Immobilize ligand''' | #'''Immobilize ligand''' | ||
#*Centrifuge Coupling Gel (5-10 minutes at 1000xg) and remove ligands solution. | #*Centrifuge Coupling Gel (5-10 minutes at 1000xg) and remove ligands solution. | ||
Revision as of 12:53, 18 August 2006
Before you begin, you will need at least 1mg of protein (eg.GST-FAA-N) and at least 1mg of ‘clearing’ protein (probably GST). This means that you will probably need 2 columns and twice as much reagent. The clearing protein will be used to make its own column in the exact same way as the fusion protein column. If you are using peptide for this column you will skip step A.
- Buffer exchange
- Dilute ligand (eg. FAA-N-GST) 1:10 in pH 10 Coupling Buffer. Reconcentrate to 1ml in 50mL 10Kmwco Centricon
- Make 1:3mL dilution of exchanged sample. This is the Ligand Solution.
- make slurry
- Put 2-5mL of Coupling Gel into a 14mL conical centrifuge tube.
- Wash w/ 5mL Coupling Buffer pH 10. Centrifuge for 5-10minutes at 1000xg. Aspirate supernate carefully.
- Add Ligand Solution. Rock for 4hours at 4∞C.
- Immobilize ligand
- Centrifuge Coupling Gel (5-10 minutes at 1000xg) and remove ligands solution.
- Wash coupling gel w/5mL of coupling buffer pH7.2.
- Make up 5M Cyanoborohydride solution. (.0785g/250µL dH20). Make solution in the hood and make the solution fresh each time you use it. Check and make sure the solution is white or whitish-yellow (Sigma).
- Combine 2mL Coupling Buffer pH7.2 and 40µL of 5M Cyanoborohydride. Rock overnight at room temperature.
