GFP纳米抗体偶联agarose beads（affinity gel）_详细资料

产品信息

保存条件： 4度浓度： 50% slurry 应用范围： Immunoprecipitation (IP), 20μL of gel slurry. 2 μg of GFP can be precipitated with 10 μL of 50% gel slurry. Optimal working dilutions must be determined by end user. 形态：液体亚型： VHH, Single Domain antibody 免疫原： GFP purified from GFP expressed 293T cells by affinity chromatographic technique using mouse anti-GFP 适应物种： all species 宿主：羊驼标记物：琼脂糖微球抗体名： GFP标签抗体偶联agarose beads（affinity gel）抗体英文名： Anti-GFP tag nanobody (VHH, Single Domain antibody) conjugated Agarose Beads - ChIP Grade 克隆性：单克隆靶点： GFP标签融合蛋白规格： 1mL

Anti-GFP tag nanobody conjugated Agarose Beads - ChIP Grade
This product is only for research applications, not for diagnostic or therapeutic use.

Catalog Number: AT1765 Size：1 mL (50% suspension) Store at: 4°C

Background
Since the detection of intracellular Aequorea victoria Green Fluorescent Protein (GFP) requires only irradiation by UV or blue light, it provides an excellent means for monitoring gene expression and protein localization in living cells. Agarose conjugated anti-GFP nanobody can detect GFP fusion protein on immunoprecipitation.

Form of Antibody: Agarose beads Conjugated anti-GFP Nanobody, in phosphate buffered saline (PBS), pH 7.3 (+/- 0.1), 0.02% sodium azide as preservative.
50% suspension, total beads suspension volume of 1000 µL (500 µL packed beads)

Bead size: ~ 90 µm (cross-linked 8% agarose beads)

Specificity: This antibody detects GFP, YFP, and CFP-tagged proteins exogenously expressed in cells. This antibody does not detect RFP-tagged proteins. Please note that the GFP, YFP, and CFP tags add approximately 27 kDa to the molecular weight of the fusion protein.

UniProt ID: P42212

Reactivity: All Species Expected

Tested applications: Immunoprecipitation (IP),
Co-Immunoprecipitation (Co-IP),
Chromatin Immunoprecipitation (ChIP)

In IP, 25 µL of beads suspension for ~1000 µg of crude total protein solution.
Optimal dilutions/concentrations should be determined by the end user.

Binding capacity: 10 µg of GFP can be precipitated with 25 µL of beads suspension.

Host: Alpaca

Clonality: Monoclonal, nanobody

Isotype: Alpaca single domain antibody (nanobody, VHH)

Immunogen: Fusion protein corresponding to Aequorea victoria GFP aa 1-246.

Positive Controls: exogenously expressed GFP proteins in cells or E. coli.

Storage instruction: Store at +4°C and do not freeze.

Expiration Date: Expires one year from date of receipt when stored as instructed

Attention: Agarose Beads should be resuspended well before used in IP, Co-IP or ChIP.

Protocol for Immunoprecipitation (IP) of GFP-Fusion Proteins from mammalian cell extract

1. Harvest cells
1). For one immunoprecipitation reaction the use of ~10⁶ - 10⁷ mammalian cells (approx. one 10-cm dish) expressing a GFP-tagged protein of interest is recommended. To harvest adherent cells, aspirate growth medium, add 1 mL ice-cold PBS to cells and scrape cells from dish. Transfer cells to a pre-cooled tube, spin at 500 g for 3 min at +4°C and discard supernatant. Wash cell pellet twice with ice-cold PBS, gently resuspending the cells.

2. Lyse cells
1). Resuspend cell pellet in 200 µL ice-cold lysis buffer by pipetting or using a syringe.
note: Supplement lysis buffer with protease inhibitors and 1 mM PMSF (not included).
optional for nuclear/chromatin proteins: Use RIPA buffer supplemented with 1 mg/mL DNase, 2.5 mM MgCl2, protease inhibitors and 1 mM PMSF (not included).

2). Place the tube on ice for 30 min with extensively pipetting every 10 min.

3). Centrifuge cell lysate at 20000x g for 10 min at +4°C. Transfer lysate to a pre-cooled tube. Add 300 µL dilution buffer to lysate. Discard pellet.
note: At this point cell lysate may be put at -80°C for long-term storage.
optional: Add 1 mM PMSF and protease inhibitors (not included) to dilution buffer.

3. Equilibrate beads
1). Vortex anti-GFP agarose beads and pipette 25 µL bead slurry into 500 µL ice-cold dilution buffer. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.

4. Bind proteins
1). Add diluted lysate (step 2.3) to equilibrated anti-GFP agarose beads (step 3.1). If required, save 50 µL of diluted lysate for immunoblot analysis. Tumble end-over-end for 1 hour at 4°C.

2). Centrifuge at 2500x g for 2 min at +4°C. If required, save 50 µL supernatant for immunoblot analysis. Discard remaining supernatant.

5. Wash beads
1). Resuspend anti-GFP agarose beads in 500 µL ice-cold dilution buffer. Centrifuge at 2500x g for 2 min at +4°C. Discard supernatant and repeat wash twice.
optional: Increase salt concentration in the second washing step up to 500 mM.

6. Elute proteins
1). Resuspend anti-GFP agarose beads in 100 µL 2x SDS-sample buffer.

2). Boil resuspended anti-GFP agarose beads for 10 min at 95°C to dissociate immunocomplexes from anti-GFP agarose beads. anti-GFP agarose beads can be collected by centrifugation at 2500x g for 2 min at 4°C and SDS-PAGE is performed with the supernatant.

3). optional instead of steps 6.1 and 6.2: elute bound proteins by adding 50 µL 0.2 M glycine pH 2.5 (incubation time: 30 sec under constant mixing) followed by centrifugation. Transfer the supernatant to a new tube and add 5 µL 1M Tris base pH 10.4 for neutralization. To increase elution efficiency this step can be repeated.

Suggested buffer composition

Buffer Composition

Lysis buffer (Co-IP) 10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.5% NP-40

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

Dilution/Wash buffer 10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA

Glycine-elution buffer 200 mM glycine pH 2.5

2 x SDS-sample buffer 120 mM Tris/Cl pH 6.8; 20% glycerol; 4% SDS, 0.04% bromophenol blue; 10% β-mercaptoethanol

Related products

Product Cat. no.

Anti-Myc tag Mouse mAb AT0023

Anti-HA tag Mouse mAb AT0024

Anti-His tag Mouse mAb AT0025

Anti-GST tag Mouse mAb AT0027

Anti-GFP tag Mouse mAb AT0028

Anti-V5 tag Mouse mAb AT0026

Anti-RFP tag Mouse mAb AT0038

Protein A/G Agarose Beads for IP/CoIP IF0001

Protein A/G Magnetic Beads for IP/CoIP IF0002

For research use only. Not for use in diagnostic or therapeutic procedures.

Engibody Biotechnology, Inc. | 8 The Green STE A, Dover, DE. 19901, USA | www.engibody.com

©2019 Engibody Biotechnology, Inc. All rights reserved. The trademarks mentioned herein are the property of Engibody Biotechnology, Inc. and/or its affiliates or their respective owners.

Version 2018-12-18

For the purchase of this product visit www.engibody.com Online ordering or email order@engibody.com
For support please email support@engibody.com

Citation Tips
If you use this product in any your published paper, please cite:
Engibody Biotechnology, Inc.
Dover, DE. 19901, USA

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Form of Antibody:	Agarose beads Conjugated anti-GFP Nanobody, in phosphate buffered saline (PBS), pH 7.3 (+/- 0.1), 0.02% sodium azide as preservative. 50% suspension, total beads suspension volume of 1000 µL (500 µL packed beads)
Bead size:	~ 90 µm (cross-linked 8% agarose beads)
Specificity:	This antibody detects GFP, YFP, and CFP-tagged proteins exogenously expressed in cells. This antibody does not detect RFP-tagged proteins. Please note that the GFP, YFP, and CFP tags add approximately 27 kDa to the molecular weight of the fusion protein.
UniProt ID:	P42212
Reactivity:	All Species Expected
Tested applications:	Immunoprecipitation (IP), Co-Immunoprecipitation (Co-IP), Chromatin Immunoprecipitation (ChIP) In IP, 25 µL of beads suspension for ~1000 µg of crude total protein solution. Optimal dilutions/concentrations should be determined by the end user.
Binding capacity:	10 µg of GFP can be precipitated with 25 µL of beads suspension.
Host:	Alpaca
Clonality:	Monoclonal, nanobody
Isotype:	Alpaca single domain antibody (nanobody, VHH)
Immunogen:	Fusion protein corresponding to Aequorea victoria GFP aa 1-246.
Positive Controls:	exogenously expressed GFP proteins in cells or E. coli.
Storage instruction:	Store at +4°C and do not freeze.
Expiration Date:	Expires one year from date of receipt when stored as instructed

Buffer	Composition
Lysis buffer (Co-IP)	10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA; 0.5% NP-40
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
Dilution/Wash buffer	10 mM Tris/Cl pH 7.5; 150 mM NaCl; 0.5 mM EDTA
Glycine-elution buffer	200 mM glycine pH 2.5
2 x SDS-sample buffer	120 mM Tris/Cl pH 6.8; 20% glycerol; 4% SDS, 0.04% bromophenol blue; 10% β-mercaptoethanol

Product	Cat. no.
Anti-Myc tag Mouse mAb	AT0023
Anti-HA tag Mouse mAb	AT0024
Anti-His tag Mouse mAb	AT0025
Anti-GST tag Mouse mAb	AT0027
Anti-GFP tag Mouse mAb	AT0028
Anti-V5 tag Mouse mAb	AT0026
Anti-RFP tag Mouse mAb	AT0038
Protein A/G Agarose Beads for IP/CoIP	IF0001
Protein A/G Magnetic Beads for IP/CoIP	IF0002