Jul 25, 2023
Co-immunoprecipitation using GFP-trap
- Dan Dou1,
- Erika Holzbaur1
- 1University of Pennsylvania
Protocol Citation: Dan Dou, Erika Holzbaur 2023. Co-immunoprecipitation using GFP-trap. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr36o2vmk/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: July 20, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 85310
Keywords: ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000350
Abstract
Here, we describe performing co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads
See "Protocol: HEK293 cell culture for co-immunoprecipitation experiments" for preceding culture and transfection. For the present study, HEK cells were transfected with EGFP-tagged bait proteins. Prey proteins were either endogenous or transfected with HA- or SNAP-tags.
Prepare wash buffer:
| A | B | |
| Tris-HCl pH 7.5 | 10 mM | |
| NaCl | 150 mM | |
| EDTA | 0.5 mM | |
| Triton-X (optional) | 0.4% |
Note
Triton-X should be included for prey proteins where appreciable non-specific binding is observed in the EGFP vector condition.
Prepare lysis buffer. Lysis buffer composition differed for experiments needing lambda phosphatase treatment
Lysis buffer for non-lambda phosphatase experiment:
| A | B | |
| Tris-HCl pH 7.5 | 10 mM | |
| NaCl | 150 mM | |
| EDTA | 0.5 mM | |
| NP-40 | 0.5% | |
| PMSF | 1 mM | |
| TAME | 0.01 mg/mL | |
| Leupeptin | 0.01 mg/mL | |
| Pepstatin A | 0.001 mg/mL |
Lysis buffer compatible with lambda phosphatase experiment:
| A | B | |
| 1x NEBuffer for Protein MettaloPhosphatases (New England BioLabs) | 1x | |
| NP-40 | 0.5% | |
| Leupeptin | 0.01 mg/mL | |
| ddH2O | To desired volume |
24 hours after transfection, wash HEK cells twice in ice-cold PBS and lyse in appropriate lysis buffer. Use 600 µL lysis buffer per experimental condition (pooled across the three 10cm dishes).
Clarify lysates at 10 x g at 4 degrees C for 10 minutes
Wash 25 µL GFP-trap beads per experimental condition in 500 µL wash buffer, in low protein binding Eppendorf tubes under rotating agitation.
Note
For large protein complexes, GFP-Trap Magnetic Particles M-270 should be used instead of GFP-Trap Magnetic Agarose beads
Equilbrate beads in lysis buffer for 5 min at 4 degrees C under rotating agitation
For lambda phosphatase experiments: add 60 µL of 10 mM MnCl2 and 24 µL lambda phosphatase (2,400 units; New England BioLabs) for a final reaction volume of 600 µL per experimental condition.
Note
For conditions that are lambda phosphatase-negative, add 24 µL ddH2O instead
Incubate at 30 degrees C for 30 minutes
Incubate beads with clarified lysate (or lambda phosphatase-treated lysate) for 1 hour at 4 degrees C under rotating agitation
Wash beads three times for 5 min in wash buffer at 4 degrees C under rotating agitation
Resuspend beads in 60 µL denaturing buffer, and boil for 10 minutes to release bound protein