Feb 06, 2025
Protocol for the production of RAB1A-10xHis in HEK293Gnti cells
- 1University of California, Berkeley
Protocol Citation: Annan SI Cook 2025. Protocol for the production of RAB1A-10xHis in HEK293Gnti cells. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9be5lk5/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: February 05, 2025
Last Modified: February 06, 2025
Protocol Integer ID: 119639
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000350
Abstract
Protocol for production of RAB1A-10xHis
Materials
Cell culture and transfection:
HEK 293T GNTi cells
pCAG RAB1A-10xHis DNA
Polyethyleneimine (PEI) (Polysciences) 20,000 Da
Hybridoma media (Thermo)
Freestyle Media
Buffers and chemicals:
Phosphate-buffered saline (PBS)
Lysis buffer:25 mM HEPES, pH 7.5, 200 mM NaCl, 2 mM MgCl₂ ,25 mM tris(2-carboxyethyl)phosphine (TCEP), 10% Glycerol
Protease inhibitor (EDTA-Free, Thermo Scientific)
Triton X-100
imidazole
Gel filtration buffer:25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1 mM TCEP
Equipment:
Bucket Centrifuge
Ultracentrifuge
Pyrex Dounce homogenizer
Rocker
NiNTA resin (2 mL)
S75 10/300 column
Liquid nitrogen
Cell growth and transfection
Cell growth and transfection
Grow 400 mL of HEK293T GNTi cells to a density of 2.0–2.2 x 10⁶ cells/mL.
Transfect the cells with 400 µg of pCAG RAB1A-10xHis construct using a four fold molar excess of PEI in 80 mL Hybridoma
Cell harvesting
Cell harvesting
After 48 hours, pellet the cells by centrifugation at 2200 rpm.
Wash and gently resuspend the pellet with PBS, centrifuge again at 500 x g for 10 minutes, and flash-freeze the pellet in liquid nitrogen. Store at -80°C.
Purification
Purification
Thaw the cell pellet at room temperature.
Resuspend the pellet in lysis buffer (25 mM HEPES (pH 7.5), 200 mM NaCl, 2 mM MgCl₂, 1 mM TCEP, 10% Glycerol) and add an EDTA-free protease inhibitor tablet (Thermo Fisher).
Transfer the resuspended pellet to a Pyrex Dounce homogenizer and homogenize the cells with 30 strokes.
Add Triton X-100 to the homogenate for a final concentration of 1%.
Gently mix and rock the mixture at 4°C for 1 hour.
Centrifuge the lysate at 17,000 rpm for 45 minutes at 4°C.
Apply the supernatant to 2 mL of NiNTA resin, repeating the application 3 times.
Wash the resin until the wash is free of protein, as determined by a Bradford assay.
Elute the protein using wash buffer supplemented with 300 mM imidazole.
Concentrate the protein and apply it to an S75 10/300 column equilibrated with buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM MgCl₂, 1 mM TCEP).
Concentrate the protein to 50 µM.
Aliquot 50 µL of the purified protein and flash freeze in liquid nitrogen.