Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant
Published: April 30, 2023
Abstract
Source: Nasimuzzaman, M. et al., Production and Purification of Baculovirus for Gene Therapy Application. J. Vis. Exp. (2018)
In this video, we demonstrate the technique to isolate baculovirus from an insect cell supernatant using heparin affinity chromatography. The isolation is facilitated by the affinity between the heparin in the column to the baculovirus envelope glycoprotein.
Protocol
1. Preparation of the Chromatography System Prepare the chromatography system by sequentially rinsing the sample and buffer lines each with sterile water, 1 N sodium hydroxide, water, and wash buffer (20 mM sodium phosphate buffer containing 150 mM sodium chloride, pH 7.0) at a linear flow rate of 50 mL/min. Prepare a heparin 50 µm column (10 × 10 cm, 7.9 mL of column volume (CV)) by sequentially running column cleaning buffer (5 CV sterile 20 mM sodium phosphate buffer containing 2 M sodi…
Divulgazioni
The authors have nothing to disclose.
Materials
| Akta Avant 150 | GE Healthcare | 28976337 | Chromatography system |
| POROS Heparin 50 µm Column | ThermoFisher Scientific | 4333414 | Heparin column |
| Wash buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 150 mmol/l sodium chloride |
| Elution buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 1.5 mol/l sodium chloride |
| 50 ml Conical tube | ThermoFisher Scientific | 14-959-49A | For collection of Baculovirus supernatant |
| Column cleaning buffer | In-house | Non-catalog item | 20 mmol/l phosphate buffer containing 2.0 mol/l sodium chloride |
| Sterile water | In-house | Non-catalog item | For Akta Avant cleaning |
| Sodium hydroxide | Sigma-Aldrich | 1.09137 | For Akta Avant cleaning |
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Citazione di questo articolo
Heparin Affinity Chromatography-Based Baculovirus Purification: A Technique to Isolate Baculovirus From Insect Cell Supernatant. J. Vis. Exp. (Pending Publication), e21095, doi: (2023).