Home | Achievement | Programmes | Projects | Experts | Staffs | Publications | Journals |
Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |

MOLECULAR BIOLOGY: WORKING WITH DNA

PURIFICATION: PROTEINS

Chromatographic Purification of Neurotrophins

Chromatographic Purification of Neurotrophins Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

Procedure Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

A. Ion-Exchange Chromatography 1. Adjust the pH of the conditioned medium to 6.5 (this is the same as IE Buffer A). (cell culture medium conditioned by the presence of cells in culture for 1 to several days, depending on cell type) 2. Equilibrate the S-Sepharose column with 6 column volumes of IE Buffer A at an 8 ml/min flow rate. 3. Apply the conditioned medium through the column at a rate of 8 ml/min. 4. Wash column with 6 column volumes of IE Buffer A (8 ml/min). 5. Elute the protein with a 0 to 1 M NaCl (IE Buffer A to IE Buffer B) linear gradient and collect 10 ml fractions (elute at 8 ml/min). 6. Identify positive fractions by western blot (see appropriate Western Blotting protocol). 7. Pool and concentrate position fractions using a centriprep-10 ultrafilter for the subsequent size exclusion chromatography. 8. Transfer protein concentrate to 1.5 ml microcentrifuge tube. B. Size Exclusion Chromatography 1. Equilibrate Uperdex-75 column with 1 column volume SE Buffer. 2. Inject protein concentrate onto the column. 3. Collect fractions. 4. Identify positive fractions by western blot (see appropriate Western Blotting protocol). 5. Pool and concentrate position fractions using a centriprep-10 ultrafilter for the subsequent size exclusion chromatography. 6. Transfer protein concentrate to 1.5 ml microcentrifuge tube. 7. Adjust the pH of the concentrated sample to around 3.5 using concentrated Acetic Acid (Glacial Acetic Acid). C. Reverse-Phase HPLC 1. Microcentrifuge the pH adjusted sample for 2 min at maximum speed to clear any insoluble material. 2. Save the supernatant for HPLC. 3. Equilibrate the C-18 HPLC column with 3 column volumes of RP Buffer A (See Protocol for general HPLC use). 4. Inject the sample and elute with 0 to 60 % acetonitrile linear gradient (RP Buffer A to RP Buffer B). 5. Identify positive fractions by western blot (see appropriate Western Blotting protocol). 6. Pool and concentrate position fractions using a centriprep-10 ultrafilter for the subsequent size exclusion chromatography. 7. Transfer protein concentrate to 1.5 ml microcentrifuge tube. 8. Vacuum dry sample and resuspend in dd H2O.

Solutions Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

RP Buffer A    0.1 % (v/v) Trifluoroacetic Acid (CAUTION See Hint #1) Prepared in Distilled Deionized Water (dd H2O) SE Buffer    10 % (v/v) Acetonitrile (CAUTION, See Hint #1) 0.5 M NaCl 5 mM EDTA 50 mM Phosphate Buffer, pH 7.4 IE Buffer B    50 mM Phosphate Buffer, pH 6.5 5 mM EDTA 1 M NaCl IE Buffer A    50 mM Phosphate Buffer, pH 6.5 5 mM EDTA Phosphate Buffer 0.5 M pH 7.4    226 mM NaH2PO4 774 mM Na2HPO4 Phosphate Buffer 0.5 M pH 6.5    700 mM Sodium Phosphate Monobasic (NaH2PO4) 300 mM Sodium Phosphate Dibasic (Na2HPO4) RP Buffer C    Prepared in ddH2O 70 % Acetonitrile RP Buffer B    Prepared in Acetonitrile 0.1 % (v/v) Trifluoroacetic Acid

BioReagents and Chemicals Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

S-Sepharose Sodium Phosphate Monobasic Acetonitrile EDTA Sodium Chloride Glacial Acetic Acid Trifluoroacetic Acid Sodium Phosphate, Dibasic Superdex-75

Protocol Hints Title | Procedure | Solutions | BioChemicals | Hints | Printable Version

1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.