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GFP-multiTrap®

ABIN1558271 产品详细信息, 供应商: Log in to see
抗原
  • green fluorescent protein
  • gfp
适用
Aequorea victoria
11
宿主
Camelidae
抗体类型
Recombinant Antibody
实验类型
Sandwich ELISA
应用范围
Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Enzyme Activity Assay (EAA), Immunoprecipitation (IP), Mass Spectrometry (MS), Protein Complex Immunoprecipitation (Co-IP), Pull-Down Assay (Pull-Down), Purification (Purif)
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原理 GFP-Trap® immobilized in microplate wells to test GFP fusion proteins for peptide, protein, DNA or RNA binding.
品牌 GFP-Multitrap®,GFP-Trap®
样品类型 Cell Extracts
Analytical Method Quantitative
检测方法 Fluorometric
特异性 Binding capacity: 1 µg GFP / well
交叉反应 (详细) GFP-Trap® specifically binds to eGFP, wtGFP, GFP S65T, TagGFP, eYFP, YFP, Venus, Citrin, CFP. No binding to proteins derived from DsRed, all RFPs and TurboGFP can be detected.
产品特性 You’ve got more than just a few samples to analyze? Then take advantage of the proven efficiency of our GFP-Trap® in a convenient 96-multiwell format.
As the GFP-Trap® is immobilized in the wells no centrifugation is necessary and you can easily test your GFP fusion proteins for peptide, protein, DNA or RNA binding.
Rapidly quantify your input, wash and bound fractions of GFP fusion proteins and fluorescently labeled binding substrates with fluorescence scanners and plate readers.
The green fluorescent protein (GFP) and variants thereof are widely used to study the subcellular localization and dynamics of proteins. GFP fusion proteins can be expressed in different cell typesat different expression levels by transient or stable transfection. Transient expression may provide quick informative results, however, in many cases it is necessary to generate stable cell lines that express the GFP fusion protein of interest at a level similar to the one of the endogenous protein. Quantification of GFP fusion proteins in cells can be tricky since existing methods, like fluorescence microscopy or Western Blotting, are often shows insufficient signal to noise ratios or high signal variabilities . The major challenge is to increase the sensitivity while keeping the background low. The following protocol describes the accurate quantification of GFP fusion proteins in cellular extracts using a new Sandwich ELISA comprising the highly sensitive GFP-multiTrap® in combination with a highly sensitive monoclonal GFP antibody.
组件 GFP-Trap® immobilized in wells
别名 GFP
背景 The green fluorescent protein (GFP) and variants thereof are widely used to study the subcellular localization and dynamics of proteins. GFP fusion proteins can be expressed in different cell types at different expression levels by transient or stable transfection. Transient expression may provide quick informative results, however, in many cases it is necessary to generate stable cell lines that express the GFP fusion protein of interest at a level similar to the one of the endogenous protein. Quantification of GFP fusion proteins in cells can be tricky since existing methods, like fluorescence microscopy or Western Blotting, are often shows insufficient signal to noise ratios or high signal variabilities .
研究领域 Tags/Labels
应用备注 Tested applications:
  • protein-protein interactions
  • protein-DNA interactions
  • protein-histone tail peptides interactions
说明

Highlights of GFP-multiTrap

  • Fast and easy capture of GFP-tagged proteins and complexes
  • High Throughput Analysis of protein interactions (incl. DNA, RNA or peptide binding)
  • No centrifugation steps
  • No unspecific binding
  • No denaturing of the protein upon binding
  • Pre-blocked

实验时间 1.5 h
板类型 Pre-coated
实验流程
  • Robust and versatile tool for biochemical analyses of GFP-fusion proteins
  • Short incubation times (5 - 30 min)
  • Quantitative isolation of fusion proteins and transiently bound factors from cell extracts or organelles
  • Low unspecific binding
  • No contaminating heavy and light chains of conventional antibodies
  • Applicable in Chromatin Immunoprecipitation (ChIP)
试剂准备

Suggested Buffers (as tested in our laboratory)

Lysis-buffer (for IP): 10 mM Tris/Cl pH7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP40, 1 mM PMSF has to be freshly added, 1x Protease Inhibitor Cocktail (e.g. Serva®) has to be freshly added, DNaseI final conc. 1 µg/µl, 2.5 mM MgCl2
Dilution-buffer: 10 mM Tris/Cl pH7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM PMSF has to be freshly added (optional), 1x Protease Inhibitor Cocktail (e.g. Serva®) has to be freshly added

样品收集
  • 1. Resuspend cell pellet (~10^7 cells) in 100 µL lysis buffer by pipetting
  • 2. Place the tube on ice for 30 min with extensively pipetting every 10 min or / and sonify 5x 0,2 sec, 2 sec break
  • 3. Spin cell lysate at 20.000x g for 10 minutes at 4°C
  • 4. Transfer supernatant to a pre cooled tube and discard pellet
  • 5. Add 400 µl dilution buffer
  • 6. The cell lysate can be frozen at this point for long-term storage at minus 80°C
  • 7. Prepare serial dilution of the cell extract in phosphate buffered saline (PBS)
实验流程

The major challenge is to increase the sensitivity while keeping the background low. The following protocol describes the accurate quantification of GFP fusion proteins in cellular extracts using a new Sandwich ELISA comprising the highly sensitive GFP-multiTrap® in combination with a highly sensitive monoclonal anti-GFP antibody.

Sandwich-ELISA

  • 8. Add 100 µL of diluted cell extract to each well of the microtiter plate and incubate for 1h at RT
  • 9. Wash the microtiter plate twice with PBS, 300 µL/well
  • 10. Block the microtiter plate by adding 300 µL 1 wt.% milk in PBS (MPBS) to each well. Incubate for 1h at RT
  • 11. Add 100 µL anti-GFP-antibody (3E5, ChromoTek) at 5 µg/mL in 5 wt.% MPBS to each well and incubate 1h at RT
  • 12. Wash the microtiter plate three times with PBS 0.05% Tween-20 (PBST) and three times with PBS, 300 µL/well
  • 13. Add 100 µL detection antibody (e.g. anti-rat-HRP-antibody) at 0.4 µg/mL in 5 wt.% MPBS to each well and incubate for 1h at RT
  • 14. Wash three times with PBST followed by three washing steps with PBS, 300 µL/well
  • 15. Add 100 µL 3,3′,5,5′-tetramethylbenzidine solution to each well and incubate 15 – 30 minutes at RT
  • 16. Stop the reaction by adding 100 µL 2M Sulfuric Acid to each well
  • 17. Measure the absorbance of each well at 450 nm in a photometer

限制 仅限研究用
注意事项 Do not freeze.
储存条件 4 °C
有效期 12 months
厂商提供的图像
Western Blotting (WB) image for GFP-multiTrap® (ABIN1558271) Comparison of GFP-Trap® with conventional mono- and polyclonal antibodies Immunopreci...
 image for GFP-multiTrap® (ABIN1558271) Comparison of GFP-Trap_A and GFP-Trap_M Left (IP): Pulldown of GFP with GFP-Trap_A an...
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