Feb 02, 2024
Very low-density lipoprotein receptor (VLDLR)-C-tag purification from HEK293E cells
- Andreas Bracher1,
- Patricia Yuste-Checa1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Andreas Bracher, Patricia Yuste-Checa, F Ulrich Hartl 2024. Very low-density lipoprotein receptor (VLDLR)-C-tag purification from HEK293E cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkoqw1v5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 25, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94601
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to purify recombinant Very low-density lipoprotein receptor (VLDLR)-C-tag protein from HEK293E cells.
Attachments
Materials
Buffers
- Binding buffer:
| A | B | |
| Tris-HCl pH 7.2 | 20 mM | |
| NaCl | 100 mM | |
| CaCl2 | 0.5 mM |
- Elution buffer:
| A | B | |
| Tris-HCl pH 7.0 | 20 mM | |
| MgCl2 | 2 M | |
| CaCl2 | 2 mM |
FreeStyle™ 293 Expression MediumThermo FisherCatalog #12338018
CaptureSelect™ tPA Affinity MatrixThermo FisherCatalog #2943430005
Amersham NAP-25 ColumnsCytivaCatalog #17-0852-01
VLDLR-C-tag expression
VLDLR-C-tag expression
Express VLDLR-C-tag in HEK293E cells cultured in FreeStyle 293 Expression Medium for 96:00:00
4d
Centrifuge culture and keep conditioned medium.
Dialyze 300 mL conditioned medium Overnight against 10 L Binding buffer.
CaptureSelect C-tag affinity chromatography
CaptureSelect C-tag affinity chromatography
Load dialyzed conditioned medium onto a CaptureSelect C-tag affinity column previously equilibrated with binding buffer (column volume, CV: 4 mL slurry for 300 mL dialyzed media) by gravity flow at 4 °C .
Wash the column with 5 CV of Binding buffer.
Elute VLDLR-C-tag protein with 6x 1 mL of Elution buffer. Collect fractions of 1 mL .
Analyze eluted fraction by SDS-PAGE and Coomassie blue staining.
Pool fractions containing VLDLR-C-tag protein.
Exchange protein buffer to Binding buffer with a NAP-25 desalting column previously equilibrated with Binding buffer.
Collect the fractions containing protein, concentrate by ultrafiltration to >1 mg mL-1, aliquot and flash-freeze purified VLDLR-C-tag in liquid nitrogen for storage at -70 °C .
Note
Approximate yield: From 300 ml of conditioned media around 0.6 mg of pure VLDLR-C-tag were obtained. Yield can be significantly increased if the VLDLR chaperone, RAP is co-overexpressed during protein production.