Jul 10, 2024
Coimmunoprecipitation
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Coimmunoprecipitation. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn6m8pl5d/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 10, 2024
Last Modified: July 10, 2024
Protocol Integer ID: 103175
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Co-Immunoprecipitation assays in HEK cells
**Transfect HEK293T Cells** - Transfect HEK293T cells with Ezrin/Atg7 cDNA using X-tremeGENE HP (Roche) 36 hours prior to lysis. - Grow cells to 85-90% confluency.
**Collect Cells for Lysis** - At 36 hours post-transfection, rinse cells with 1x PBS. - Collect cells for lysis.
**Lysis and Protein Extraction** - Briefly vortex cells in chilled membrane solubilization buffer (25 mM HEPES, 150 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 10% Glycerol) with protease (cOmplete, Roche) and phosphatase (PhosSTOP, Roche) inhibitors.
**Equalize Protein Concentrations** - Use the Pierce™ BSA Protein Assay Kit (Thermo Fisher) and CLARIOstar Plus Plate Reader (BMG Labtech) to determine protein concentrations. - Equalize cell lysate concentrations.
**Incubate with Magnetic Beads** - Incubate equalized cell lysates with Pierce™ Anti-c-Myc Magnetic Beads (Thermo Fisher) for 6 hours at 4°C while rotating.
**Wash Magnetic Beads** - Wash beads with chilled lysis buffer.
**Elute Protein Samples** - Elute protein samples from beads by adding 2x Bromophenol Blue-free Laemmli Sample Buffer. - Heat samples to 95°C for 5 minutes.
**Perform Western Blot Analysis** - Subject eluted protein samples to western blot analysis using a SimpleWestern Jess (ProteinSimple) automated immunoassay system with a 12-230 kDa Fluorescence Separation module and the manufacturer's protocol.
**Primary Antibodies for Detection** - Use the following primary antibodies: - Anti-Myc (Rabbit, 1:40, Cell Signaling Technologies, mAb#2278) - Anti-HA (Rat, 1:20, Roche, 11867423001)
**Secondary Antibodies for Signal Detection** - Use the following fluorescently conjugated secondary antibodies at a 1:100 dilution: - IRDye® 680RD Goat anti-Rabbit IgG (LI-COR, 926-68071) - IRDye® 680RD Goat anti-Rat IgG (LI-COR, 926-68076)
**Quantify Protein Signals** - Quantify protein signals using the Simple Western Compass software. - Utilize quantitative electropherograms of detected signals. - Background-subtract and normalize Ezrin co-immunoprecipitation signal intensity to Ezrin protein load and Atg7 IP levels for statistical analysis.
**Statistical Analysis** - Perform statistical analysis in GraphPad Prism 9. - Use One-way ANOVA with Tukey’s multiple comparisons test. - Set alpha threshold to 0.05.