Jan 18, 2023
Immunoprecipitation from transfected cells
- 1University of Rochester
Protocol Citation: Cesare Orlandi 2023. Immunoprecipitation from transfected cells. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l27353g1y/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 18, 2023
Last Modified: January 18, 2023
Protocol Integer ID: 75491
Abstract
This protocol provides step by step information on how to perform immunoprecipitation from transfected cells. We normally use this protocol with HEK293FT cells but it can be adopted to other cell types.
Guidelines
Note: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.
Materials
IP buffer:
Substances Final conc. (/50 ml)
H2O (ice-cold) N/A 38.25 ml
1 M Tris-HCl (pH 7.4 at 4°C) 50 mM 2.5 ml
4 M NaCl 300 mM 3.75 ml
10% (w/v) Triton X-100 1% 5 ml
add fresh Protease Inhibitors
Immunoprecipitation from transfected cells
Immunoprecipitation from transfected cells
Drain medium and gently wash cells once with ice-cold PBS (0.5ml for mw6; 1 ml for 6-cm dish; 3 ml for 10-cm dish).
Use a gentle stream to dislodge cells with PBS.
Centrifuge the tube at 3000 x g for 1 min and discard the supernatant.
Resuspend pellet of cells with 500 μl of ice-cold IP buffer
Lyse the cells by sonication: 3 times 10’’/30% on ice, waiting 1 minute in between for the lysate to cool down
Incubate the whole cell lysate on an end-to-end rocker at 4°C/30’ to solubilize the membrane proteins.
Spin down at 14,000 x g for 10 minutes at 4°C to pellet debris and genomic DNA, save the supernatant.
Save 75 µl, Cell Lysate (CL) and add 25 µl of SB4X
Mix total proteins (equal amounts for each sample) with 10-20 µl of Protein G beads and 2 µg of specific antibody. Note: wash Protein G Beads 3 times with PBS to remove ethanol present in the storage buffer. Let equilibrate in IP buffer for minute before use.
Incubate on an end-to-end rocker at 4°C/overnight
Separate the beads and save 75 µl of Flow Through (FT) and add 2 µl of SB4X
10. Wash beads 3 times with 1 ml of IP buffer
11. Elute with 50 µl of SB1X (IP)
12. Warm samples at 42°C/10’ (membrane proteins would start to aggregate at higher temperature).
13. Load 10 µl of each sample per well for SDS-PAGE