A: Isolate HHHHHHH
Method: Ni-Affinity Chromatography
Reason: Six histadine amino acids at the end of the protein can bind to nickel very tightly. Nickel can bind to agarose bead very tightly. Use this strong affinity column we can isolate our protein, which has seven histadine amino acids.
Buffer condition:
His-binding buffer:
• 50 mM Tris-HCl (pH8.0)
• 5 mM Imidazole
• 100 mM NaCl
• 0.1 mM EDTA
• 1 mM PMSF made fresh
His-wash buffer:
• 50 mM Tris-HCl (pH8.0)
• 300 mM NaCl
• 15 mM Imidazole
• 0.1 mM EDTA
• 1 mM PMSF made fresh
is-elution buffer:
• 50 mM Tris-Cl (pH8.0)
• 50 mM NaCl
• 300 mM Imidazole
• 0.1 mM EDTA
• 1 mM PMSF made fresh
Procedure:
1. Ni-Agarose Beads Preparation:
1L mixture proteins will need 25 ml of beads.Transfer beads into a column. Wash the column by His Elution Buffer then wash by His Binding Buffer.
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Since our protein has seven histadine amino acids and no other amino acids present so the protein should have high affinity, in this way we will use column binding instead of batch binding to purify protein. Flow the mixture through the column. Save small amount of eluate.
3. Wash beads with His Binding Buffer. Save small amount of eluate.
4. Wash the column with His Wash Buffer. Check
flow through with quick bradford. Continue with the His wash buffer until no detectible protein is found
in eluate which means all of the weakly binding proteins are washed away.
5. Elute the protein with His Elution Buffer. Save all