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6/14/2021

Application noteInhalt
1.

Introduction .................................................................................................................................1

2.

Protocol ........................................................................................................................................1

1. Introduction
Green Fluorescent Protein (GFP) has been used for decades as an invaluable tool in plant research
not only for monitoring trafficking and subcellular localization of proteins of interest but also foridentifying binding partners to the protein of interest in co-immunoprecipitations (Co-IP). This
Application Note provides a short protocol for the use of GFP-Trap® Agarose for IPs from Arabidopsis
thaliana samples. It is based on more than 70 peer reviewed publications available athttps://www.chromotek.com/references/. Please note that the protocol may also serve as inspiration
when working with other plants e.g. Nicotiana (see online references).

2. Protocol
This Application Note describes how to prepare harvested plant material for a pull-down of GFPtagged fusion proteins (and potential binding partners when doing a Co-IP) with ChromoTek’s GFPTrap Agarose. Prior to this, the target protein vector (containing the GFP-fusion protein sequence)has been transformed into plant cells using standard transformation methods. Then, thetransformed plant cell culture has been cultivated and harvested.

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IP of Arabidopsis thaliana plant samples with GFP-Trapinfo@chromotek.com
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2.1.

Cell material

The amount of sample material depends on the expression level of the GFP-tagged fusion protein.
For one IP reaction it is recommended to use 1-10 g of seedling or leaf material. Alternatively, 1-20
mg of total protein extracts can be used.

2.1.

Materials and buffer composition

This protocol describes the immunoprecipitation with GFP-Trap® Agarose (product code: gta). GFPTrap® Magnetic Agarose (product code: gtma) or GFP-Trap® Magnetic Particles M-270 (product code:gtd) can alternatively be used because they contain the same anti-GFP Nanobody as GFP-Trap
Agarose. The handling of magnetic beads would be adapted from this protocol so that separation isaccomplished by a magnet instead of centrifugation. See the corresponding manuals for additional
information.
Suggested buffer composition:
Lysis buffer

50 mM Tris/HCl pH 7.5, 150 mM NaCl OR
25 mM Hepes/KOH pH 7.4, 100 mM NaCl
supplements (depending on protein of interest):
o 10-15 mM MgCl2o 5-15% glycerol
o 0.5-2 mM EDTAo 1-10 mM DTT
o 1% Triton™ X-100 or 0.1- 0.5% Nonidet™ P-40 Substituteo 1 mM PMSF or 1x protease inhibitor cocktail, 1x phosphataseinhibitor cocktail

Wash buffersame as the lysis buffer

2x SDS-sample buffer

120 mM Tris/HCl pH 6.8, 20 % glycerol, 4 % SDS, 0.04 % bromophenol
blue, 10 % ß-mercaptoethanol

2.2.
1.

Cell lysis
Harvest plant material and grind into a fine powder in liquid nitrogen.

2. Transfer powder to a 15 mL tube.
Optional: Store in a freezer.
3.

Resuspend powder in lysis buffer: 1-3 mL lysis buffer per 1 g powder.

4. Mix by vortexing for 1 min, incubate for 30 min at +4°C / on ice.
Optional: Pass lysate through a 1 mL syringe with needle to further facilitate cell lysis.
5. Remove cell debris by centrifugation (e.g. 20.000x g 15 min, +4°C).
Optional: Clear lysate additionally by filtration.
6.

Submit cleared lysate to IP.

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IP of Arabidopsis thaliana plant samples with GFP-Trapinfo@chromotek.com
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2.3.

Immunoprecipitation with GFP-Trap

Bead equilibration
1.

Resuspend GFP-Trap Agarose beads by gently pipetting up and down. Do not vortex thebeads!

2.

Transfer bead slurry into an appropriate reaction tube.

3. It is recommended to start with 25 µL of bead slurry for one IP reaction.
If there is still GFP-tagged protein left in the flow-through fraction, increase the volume ofbead slurry to 50 µL per IP.
4.

Add 500 μL ice-cold lysis buffer.

5.

Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C and discard supernatant.

Protein binding
1. Add cleared lysate to the equilibrated beads.
If required, save 50 μL of supernatant for further analysis (input fraction).
2.

Rotate end-over-end for 1 hour at +4°C.

Washing
1. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C.
If required, save 50 μL of supernatant for further analysis (flow-through/non-bound fraction).
2.

Discard remaining supernatant.

3.

Resuspend beads in 500 μL wash buffer.

4.

Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C and discard supernatant.

5.

Repeat this step at least twice.

6.

During the last washing step, transfer the beads to a new tube.

Optional: To increase stringency of the wash buffer, test various salt concentrations e.g. 150 mM– 500 mM, and/or add a non-ionic detergent e.g. Tween 20 or Triton™ X-100.
Elution with 2x SDS-sample buffer
1.

Remove the remaining supernatant.

2.

Resuspend beads in 80 μL 2x SDS-sample buffer.

3.

Boil beads for 5 min at +95°C to dissociate immunocomplexes from beads.

4.

Sediment the beads by centrifugation at 2,500x g for 2 min at +4°C.

5.

Analyze the supernatant in SDS-PAGE and Western blot.

As an alternative to sample analysis by SDS-PAGE/WB bound proteins be investigated by mass
spectrometry after on-bead digestion.

3IP of Arabidopsis thaliana plant samples with GFP-Trap