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Protein Immunoprecipitation in Maize

María Jazmín Abraham-Juárez María Jazmín Abraham-Juárez

Published: Jun 5, 2019 DOI: 10.21769/BioProtoc.3256 Views: 3750

Edited by: Marisa Rosa

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Abstract

Protein immunoprecipitation (IP) is a method used to identify cellular protein complexes. Whence, antibodies that specifically recognize a native bait protein or an epitope tag fused to the protein of interest allow the protein of interest to be purified in complexes with other proteins. The extraction buffer must be stringent enough to dissociate weakly-interacting proteins to have a manageable number of proteins for mass spectrometry as well as to increase reproducibility between replicates, but not so stringent than can dissociate all the interactions. So, it is critical to have replicates to can identify reliable candidates to be interacting. As a model plant for grasses, it is important to have reproducible methods to analyze in vivo protein-protein interactions in maize.

Here, I describe a simple and reproducible method for immunoprecipitation of native maize proteins using specific antibodies. This method also has been tested–with excellent results–for IP using magnetic beads coupled to an Anti-GFP antibody. I show the IP of a very low abundant membrane-localized protein called NOD (NARROW ODD DWARF).

Keywords: Maize search Protein purification search Immunoprecipitation search Western blot search Mass spectrometry search

Materials and Reagents

  1. Plant tissue (3 to 5 g for CoIP and 8 g for IP to be analyzed by LC-MS/MS)
  2. Protease-free 1.5 ml Eppendorf tubes
  3. Protease-free 35 ml Sorvall centrifuge tubes
  4. Miracloth, pore size: 22-25 μm (Merck Millipore, catalog number: 475855)
  5. 0.22 μm syringe filter, Millex-GP polyethersulfone 33 mm sterilized (Sigma, catalog number: SLGP033RS)
  6. Protease-free pipette tips (1,000, 200 and 10 μl)
  7. 15 ml Falcon tubes
  8. 12 x 12 cm square Petri dish
  9. Liquid nitrogen
  10. Tris base (Sigma, catalog number: T1503)
  11. Sodium chloride (Sigma, catalog number: S7653)
  12. IGEPAL-CA-630 (Sigma, catalog number: I8896)
  13. CompleteTM Protease Inhibitor Cocktail tablets (Sigma, catalog number: 11697498001)
  14. PhosSTOPTM Phosphatase inhibitor tablets (Sigma, catalog number: 4906845001)
  15. DL-Dithiothreitol (Sigma, catalog number: 43819)
  16. Sodium dodecyl sulfate, SDS (Sigma, catalog number: 436143)
  17. Bromophenol blue (BIORAD, catalog number: 1610404)
  18. Glycerol
  19. Polyclonal Anti-NOD produced in Guinea pig (Rosa et al., 2017) (used at 1:2,000 dilution)
  20. Polyclonal Anti-Guinea pig HRP-conjugated (used at 1:3,000 dilution) produced in Goat (Thermo Fisher Scientific, catalog number: A18769)
  21. Dynabeads M270 Epoxy (Invitrogen, Catalog number: 14301)
  22. GelCode Blue Safe protein stain (Thermo Fisher Scientific, catalog number: 24594)
  23. Reagents and materials for SDS-PAGE and Western blot (described in Abraham-Juárez, 2019)
  24. MQ water
  25. Buffer 1 (see Recipes)
  26. Buffer 2 (see Recipes)
  27. Wash buffer (see Recipes)
  28. Elution buffer (see Recipes)

Equipment

  1. Sterile spatula
  2. Micropipettes (1,000, 200 and 10 μl)
  3. Sterile mortar and pestle
  4. -80 °C freezer
  5. Refrigerated centrifuge
  6. Vortex homogenizer
  7. Magnetic separator for 1.5 ml tubes (MagneSphere® Technology Magnetic Separation Stand) (Promega, catalog number: Z5342)
  8. Equipment for SDS-PAGE and Western blot

Procedure

Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant molecular biology > Protein

Molecular Biology > Protein > Protein-protein interaction

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