Protein G Purification of Antibodies
Protein G Purification of Antibodies
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Source: Contributed by Nanci Donacki |
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Reagent and Materials
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Hi-Trap Protein G Column (Pharmacia Biotech #17-0404-01)
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20 mM Sodium Phosphate Buffer, pH 7.0
1.084 g NaH2 PO4 , anhydrous
3.273 g Na2 HPO4 . 7H2 O
q.s. to 1 liter with di-H2 O
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0.1M Glycine, pH 2.8
3.75 g Glycine
1.4 ml HCl, concentrated
q.s. to 1 liter with di-H2 O
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1M Tris
141.1 g Tris base
q.s. to 1 liter with di-H2 O.
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20% Ethanol
Procedure
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Prepare the collection tubes by adding 0.1 ml of 1M Tris per ml of each fraction to be collected.
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Centrifuge or filter the sample to remove any particulates. Adjust to pH 7.0.
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Wash the column with 5 bed volumes of 20 mM Sodium Phosphate Buffer, pH 7.0.
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Apply the sample onto the column.
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Was with 5 bed volumes of 20 mM Phosphate Buffer, pH 7.0
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Elute the antibody with 1-3 bed volumes of 0.1M Glycine, collecting fractions into tubes containing 1M Tris.
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Regenerate column with 2-% Ethanol and store at 2-8o C.
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Pool fractions containing antibody and dialyze against 3 changes of PBS, at least 100 times the sample volume.
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Determine the protein concentration of the antibody. Concentrate if necessary. Store aliquots at -20o C.
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