GFP-Trap® Agarose
Product code: gta

Introduction
The ChromoTek GFP-Trap® Agarose consists of an anti-Green Fluorescent Protein (GFP) Nanobody (VHH),
which is covalently bound to agarose beads. GFP-Trap Agarose is used to immunoprecipitate GFP-fusion
proteins from cell extracts of various organisms like mammals, plants, bacteria, yeast, insects etc.

Properties

Ligand: Anti-GFP single domain antibody fragment (VHH, Nanobody)
Reactivity: Specifically binds to most common GFP derivatives (visit www.chromotek.com for a complete listof recognized GFP variants).
Binding capacity: 25-30 µg of recombinant GFP per 25 µL bead slurry
Bead size: 90 µm (cross-linked 4 % agarose beads)
Buffer compatibility: See Wash buffer compatibility table.
Storage buffer: 20 % ethanol
Storage conditions: Upon receipt store at +4°C. Do not freeze!
Stability: Stable for 1 year upon receipt.
Shipment: Shipped at ambient temperature.
RRID: AB_2631357

Version 11/06/2021

1GFP-Trap® Agarose

GFP-Trap® Agarose
Product code: gta

Suggested buffer compositions

Required buffer solutions
NEW: Update of Wash buffer components.
BufferComposition

Lysis buffer

10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5 % Nonidet™ P40 Substitute (adjust thepH at +4°C)

RIPA buffer

10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1 % SDS, 1 % Triton™ X-100, 1 %deoxycholate (adjust the pH at +4°C)

Dilution buffer

10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA (adjust the pH at +4°C)

Wash buffer

10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05 % Nonidet™ P40 Substitute, 0.5 mM EDTA (adjust thepH at +4°C)

2x SDS-sample buffer

120 mM Tris/Cl pH 6.8, 20 % glycerol, 4 % SDS, 0.04 % bromophenol blue, 10 % βmercaptoethanol

Acidic elution buffer

200 mM glycine pH 2.5 (adjust the pH at +4°C)

Neutralization buffer

1 M Tris pH 10.4 (adjust the pH at +4°C)

Note: Use your equivalent cell lysis buffer for other cell types like yeast, plants, insects, bacteria.
Note: Consider using a Wash buffer without detergent for co-immunoprecipitation.

GFP-Trap® Agarose

2Version 11/06/2021GFP-Trap® Agarose
Product code: gta

Wash buffer compatibility table

Buffer ingredientsMax. concentration

DTT

1 mM

Glycerol30 %Guanidine HCl

4M

NaCl

2M

Nonidet™ P40 Substitutetested up to 2 %

SDS

1%

TCEP

0.2 mM

Triton™ X-100

tested up to 1 %

Urea

8M

Product sizes

ProductProduct code

Size

GFP-Trap® Agarose

gta-1010 reactions (250 µL slurry)gta-2020 reactions (500 µL slurry)gta-100

100 reactions (2.5 mL slurry)gta-200

200 reactions (5 mL slurry)gta-400

400 reactions (10 mL slurry)gtak-20

20 reactions (500 µL slurry) including buffers

GFP-Trap® Agarose Kit

Version 11/06/2021

3GFP-Trap® Agarose

GFP-Trap® Agarose
Product code: gta

Protocol at a glance

GFP-Trap® Agarose

4

Version 11/06/2021

GFP-Trap® Agarose
Product code: gta

Immunoprecipitation protocol

Cell material
The following protocol describes the preparation of mammalian cell lysate!
For other type of cells, we recommend using 500 µg of cell extract and start the protocol with step Beadequilibration.

Mammalian cell lysis
Note: Harvesting of cells and cell lysis should be performed with ice-cold buffers. We strongly recommend toadd protease inhibitors to the Lysis buffer to prevent degradation of your target protein and its bindingpartners.
For one immunoprecipitation reaction, we recommend using ~106-107 cells.
1. Choice of lysis buffer:· For cytoplasmic proteins, resuspend the cell pellet in 200 µL ice-cold Lysis buffer by pipetting up anddown. Supplement Lysis buffer with protease inhibitor cocktail and 1 mM PMSF (not included).
· For nuclear/chromatin proteins, resuspend cell pellet in 200 µL ice-cold RIPA buffer supplemented
with DNaseI (f.c. 75-150 Kunitz U/mL), MgCl2 (f.c. 2.5 mM), protease inhibitor cocktail and PMSF (f.c. 1mM) (not included).
2. Place the tube on ice for 30 min and extensively pipette the suspension every 10 min.
3. Centrifuge cell lysate at 17,000x g for 10 min at +4°C. Transfer cleared lysate (supernatant) to a precooled tube and add 300 µL Dilution buffer supplemented with 1 mM PMSF and protease inhibitor
cocktail (not included). If required, save 50 µL of diluted lysate for further analysis (input fraction).

Bead equilibration
1. Resuspend the beads by gently pipetting up and down or by inverting the tube. Do not vortex the beads!
2. Transfer 25 µL of bead slurry into a 1.5 mL reaction tube.
3. Add 500 µL ice-cold Dilution buffer.
4. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C. Discard the supernatant.
Note: Alternatively, Spin columns (sct-10; -20; -50) can be used to equilibrate the beads.

Protein binding
1. Add diluted lysate to the equilibrated beads.
2. Rotate end-over-end for 1 hour at +4°C.

Version 11/06/2021

5GFP-Trap® Agarose

GFP-Trap® Agarose
Product code: gtaWashing
1. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C.
2. If required, save 50 µL of supernatant for further analysis (flow-through/non-bound fraction).
3. Discard remaining supernatant.
4. Resuspend beads in 500 µL Wash buffer.
5. Sediment the beads by centrifugation at 2,500x g for 5 min at +4°C. Discard remaining supernatant.
6. Repeat this step at least twice.
7. 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. 150-500 mM, and/or
add a non-ionic detergent e.g. Triton™ X-100 (see Wash buffer compatibility table for maximal concentrations).
Note: Alternatively, Spin columns (sct-10; -20; -50) can be used to wash the beads.

Elution with 2x SDS-sample buffer (Laemmli)
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 / Western Blot.
Note: For Western blot detection we recommend GFP antibody [3H9] (3h9-20; -100) or GFP antibody [PABG1](PABG1-20; -100) in combination with Nano-Secondary® alpaca anti-rabbit IgG, recombinant VHH, Alexa
Fluor® 488/568/647 [CTK0101, CTK0102] (srbAF488-1-10; -100; srbAF568-1-10; -100; srbAF647-1-10; -100).

Elution with Acidic elution buffer
1. Remove the remaining supernatant.
2. Add 50-100 µL Acidic elution buffer and constantly pipette up and down for 30-60 sec at +4°C or roomtemperature.
3. Sediment the beads by centrifugation at 2,500x g for 2 min at +4°C.
4. Transfer the supernatant to a new tube.
5. Immediately neutralize the eluate fraction with 5-10 µL Neutralization buffer.
6. Repeat this step at least once to increase elution efficiency.
Note: Elution at room temperature is more efficient than elution at +4°C. Prewarm buffers for elution at roomtemperature.
Note: Alternatively, Spin columns (sct-10; -20; -50) can be used to separate the beads.

GFP-Trap® Agarose

6Version 11/06/2021

GFP-Trap® Agarose
Product code: gta

Product overview and related products

GFP toolbox

Product code

GFP-Trap® Agarose

gta-10; -20; -100®

GFP-Trap Agarose Kit

gtak-20

GFP-Trap® Magnetic Agarose

gtma-10; -20; -100®

GFP-Trap Magnetic Agarose Kit

gtmak-20

GFP-Trap® Magnetic Particles M-270

gtd-10; -20; -100®

GFP-Trap Magnetic Particles M-270 Kit

gtdk-20

iST GFP-Trap Kit for IP/MS

gtak-iST-8®

GFP-Trap Multiwell Plate

gtp-96

Binding Control Agarosebab-20Binding Control Magnetic Agarose

bmab-20

Spin columns

sct-10; sct-20; sct-50

GFP VHH, recombinant binding protein

gt-250GFP VHH, biotinylated recombinant binding protein

gtb-250

EGFP, recombinant purified protein

EGFP-250

GFP antibody [3H9] (rat monoclonal)

3h9-20; -100

GFP antibody [PABG1] (rabbit polyclonal)

PABG1-20; -100®

Nano-Secondary alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, AlexaFluor® 488 [CTK0101, CTK0102]
Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, AlexaFluor® 568 [CTK0101, CTK0102]
Nano-Secondary® alpaca anti-human IgG/anti-rabbit IgG, recombinant VHH, Alexa
Fluor® 647 [CTK0101, CTK0102]

srbAF488-1-10; -100

GFP-Booster Alexa Fluor® 488
GFP-Booster Alexa Fluor® 568
GFP-Booster Alexa Fluor® 647
GFP-Booster ATTO488
GFP-Booster ATTO594
GFP-Booster ATTO647N

gb2AF488-10; -50
gb2AF568-10; -50
gb2AF647-10; -50
gba488-10; -100
gba594-10; -100
gba647n-10; -100

srbAF568-1-10; -100
srbAF647-1-10; -100

For product details, information, and ordering visit www.chromotek.com.

Version 11/06/2021

7GFP-Trap® Agarose

GFP-Trap® Agarose
Product code: gta

Contact

support@chromotek.com

ChromoTek GmbHAm Klopferspitz 1982152 Planegg-Martinsried
Germany
phone: +49 89 124 148 80
fax: +49 89 124 148 811

ChromoTek Inc.
62-64 Enter LaneIslandia, NY 11749
USA
phone: 631 501 1058
fax: 631 501 1060

Disclaimer

Only for research applications, not for diagnostic or therapeutic use!
ChromoTek and GFP-Trap, RFP-Trap, Myc-Trap, Spot-Trap, Spot-Tag, Spot-Label, Spot-Cap, Nano-Secondary, F2H Kit, and Chromobody areregistered trademarks of ChromoTek GmbH, part of Proteintech Group. Nano-CaptureLigand and V5-Trap are trademarks of ChromoTek
GmbH, part of Proteintech Group. Nanobody is a registered trademark of Ablynx, a Sanofi company. Alexa Fluor is a registered trademark ofLife Technologies Corporation, a part of Thermo Fisher Scientific Inc. SNAP-tag is a registered trademark and CLIP-tag is a trademark of New
England Biolabs, Inc. Octet is a registered trademark of FortéBio, a Sartorius brand. Other suppliers’ products may be trademarks or registeredtrademarks of the corresponding supplier each. Statements on other suppliers’ products are given according to our best knowledge.

GFP-Trap® Agarose

8Version 11/06/2021