May 24, 2023
Immunoprecipitation (IP)
- 1Laboratory of Michael Lazarou, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia
Protocol Citation: nguyen.tha 2023. Immunoprecipitation (IP). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly79yelx9/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 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 76523
Keywords: immunoprecipitation, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000350
Abstract
This protocol details about immunoprecipitation using anti-HA magnetic beads.
Attachments
635-1314.docx
17KB
Materials
Buffers and reagents:
- IP base buffer: 50 millimolar (mM) Tris-Cl (7.5 when cold), 150 millimolar (mM) NaCl
- Bead equilibration buffer: IP base buffer supplemented with 0.1% Tween20
- IP wash buffer: IP base buffer supplemented with 0.1% TX-100 and 1x cOmplete, EDTA-free protease inhibitor cocktail
- Pierce™ Anti-HA Magnetic BeadsThermo FisherCatalog #88836
- Benzonase® NucleaseMerck MilliporeCatalog #E1014-25KU
- cOmplete™ EDTA-free Protease Inhibitor CocktailRocheCatalog #4693132001
- Elution buffer: NuPAGE™ LDS Sample Buffer (4X)Thermo Fisher ScientificCatalog #NP0007
Note
Elution buffer can be aliquoted and stored at -20 °C or -80 °C .
Procedures
Procedures
3h 50m
3h 50m
Lyse cell pellets (5-7 mg ) in 500 µL IP lysis buffer containing IP base buffer supplemented with 1x cOmplete, EDTA-free protease inhibitor cocktail and 0.1 µL of benzonase and incubate samples On ice for 00:30:00 . Mix the sample by inverting the eppies gently every 5 min.
30m
Wash anti-HA beads with 500 µL of bead equilibration buffer.
Repeat step 2 twice.
Centrifuge the cell lysates at max speed for 00:10:00 at 4 °C .
10m
Carefully transfer cleared lysates into 2 ml eppies and take 50 µL from each tube for “Input” samples.
Gently add 1000 µL of IP base buffer containing 1x cOmplete, EDTA-free protease inhibitor cocktail to the rest of each sample to dilute out the detergent.
Incubated the diluted cleared lysates with the anti-HA magenetic beads on a rotary mixer for 03:00:00 at 4 °C .
3h
Collect beads on a magnetic rack and aspirate the unbounds.
Wash with 1 mL IP wash buffer.
Repeat steps 7-8 another 4 times.
Note
For the last wash, make sure to remove all the liquid off the beads.
Elute with 25 µL elution buffer by boiling at shaking at 99 °C for 00:10:00 .
10m