GFP-multiTrap®

Details for Product No. ABIN1082196
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Target Name (Antigen)
Reactivity
Aequorea victoria
(24)
Host
Camelidae
Application
Protein Complex Immunoprecipitation (Co-IP), Mass Spectrometry (MS), Enzyme Activity Assay (EAA), Affinity Measurement (AM), Chromatin Immunoprecipitation (ChIP), Pull-Down Assay (Pull-Down), Purification (Purif), Immunoprecipitation (IP)
Pubmed 21 references available
Quantity 5 x 96 tests
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Purpose GFP-Trap® immobilized in microplate wells to test GFP fusion proteins for peptide, protein, DNA or RNA binding.
Brand GFP-Multitrap®,GFP-Trap®
Sample Type Cell Extracts
Fragment heavy chain antibody (hcAb)
Specificity Binding capacity: 1 µg GFP / well
Cross-Reactivity (Details) 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.
Characteristics 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.
Components GFP-Trap® immobilized in wells
Alternative Name GFP
Background 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 .
Research Area Tags/Labels
Application Notes Tested applications:
  • protein-protein interactions
  • protein-DNA interactions
  • protein-histone tail peptides interactions
Comment

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

Plate Black (clear bottom),96 wells
Protocol
  • 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)
Reagent Preparation 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
Sample Collection
  • 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)
Assay Procedure 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
Restrictions For Research Use only
Handling Advice Do not freeze.
Storage 4 °C
Expiry Date 12 months
Supplier Images
GFP-multiTrap® Comparison of GFP-Trap® with conventional mono- and polyclonal antibodies Immunoprec...
GFP-multiTrap® (2) Comparison of GFP-Trap_A and GFP-Trap_M Left (IP): Pulldown of GFP with GFP-Trap_A an...
Product cited in: Lahiri, Chao, Tavassoli et al.: "A Conserved Endoplasmic Reticulum Membrane Protein Complex (EMC) Facilitates Phospholipid Transfer from the ER to Mitochondria." in: PLoS biology, Vol. 12, Issue 10, pp. e1001969, 2014 (PubMed).

Montesinos, Pastor-Cantizano, Robinson et al.: "Arabidopsis p24δ5 and p24δ9 facilitate COPI-dependent Golgi-to-ER transport of the K/HDEL receptor ERD2." in: The Plant journal : for cell and molecular biology, 2014 (PubMed).

Cruz-García, López-Saavedra, Huertas: "BRCA1 Accelerates CtIP-Mediated DNA-End Resection." in: Cell reports, Vol. 9, Issue 2, pp. 451-9, 2014 (PubMed).

Webster, Colombi, Jäger et al.: "Surveillance of Nuclear Pore Complex Assembly by ESCRT-III/Vps4." in: Cell, Vol. 159, Issue 2, pp. 388-401, 2014 (PubMed).

Chen, Yeap, Bogoyevitch: "The JNK1/JNK3 interactome - Contributions by the JNK3 unique N-terminus and JNK common docking site residues." in: Biochemical and biophysical research communications, 2014 (PubMed).

Patten, Wong, Khacho et al.: "OPA1-dependent cristae modulation is essential for cellular adaptation to metabolic demand." in: The EMBO journal, 2014 (PubMed).

Shen, Ding, Gao et al.: "N-linked glycosylation of AtVSR1 is important for vacuolar protein sorting in Arabidopsis." in: The Plant journal : for cell and molecular biology, 2014 (PubMed).

Tanaka, Tan, Mochida et al.: "Hrr25 triggers selective autophagy-related pathways by phosphorylating receptor proteins." in: The Journal of cell biology, Vol. 207, Issue 1, pp. 91-105, 2014 (PubMed).

Shahid, Soroka, Kong et al.: "Structure and mechanism of action of the BRCA2 breast cancer tumor suppressor." in: Nature structural & molecular biology, Vol. 21, Issue 11, pp. 962-8, 2014 (PubMed).

De Wever, Nasa, Chamousset et al.: "The human mitotic kinesin KIF18A binds protein phosphatase 1 (PP1) through a highly conserved docking motif." in: Biochemical and biophysical research communications, 2014 (PubMed).

McGough, Steinberg, Gallon et al.: "Identification of molecular heterogeneity in SNX27-retromer-mediated endosome-to-plasma membrane recycling." in: Journal of cell science, 2014 (PubMed).

Falcone, Roman, Hnia et al.: "N-WASP is required for Amphiphysin-2/BIN1-dependent nuclear positioning and triad organization in skeletal muscle and is involved in the pathophysiology of centronuclear myopathy." in: EMBO molecular medicine, Vol. 6, Issue 11, pp. 1455-75, 2014 (PubMed).

Cobret, De Tauzia, Ferent et al.: "Targeting the Cis-Dimerization of Lingo-1 with Small-Molecule Affects Its Downstream Signaling." in: British journal of pharmacology, 2014 (PubMed).

van der Lelij, Stocsits, Ladurner et al.: "SNW1 enables sister chromatid cohesion by mediating the splicing of sororin and APC2 pre-mRNAs." in: The EMBO journal, 2014 (PubMed).

Yatsenko, Marrone, Shcherbata: "miRNA-based buffering of the cobblestone-lissencephaly-associated extracellular matrix receptor dystroglycan via its alternative 3'-UTR." in: Nature communications, Vol. 5, pp. 4906, 2014 (PubMed).

Fouquerel, Goellner, Yu et al.: "ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion." in: Cell reports, Vol. 8, Issue 6, pp. 1819-31, 2014 (PubMed).

Chan, West: "Spatial control of the GEN1 Holliday junction resolvase ensures genome stability." in: Nature communications, Vol. 5, pp. 4844, 2014 (PubMed).

Asante, Stevenson, Stephens: "Subunit composition of the human cytoplasmic dynein-2 complex." in: Journal of cell science, 2014 (PubMed).

Zhang, Liu, Ding et al.: "Fission yeast Pxd1 promotes proper DNA repair by activating Rad16XPF and inhibiting Dna2." in: PLoS biology, Vol. 12, Issue 9, pp. e1001946, 2014 (PubMed).

Blasius, Wagner, Choudhary et al.: "A quantitative 14-3-3 interaction screen connects the nuclear exosome targeting complex to the DNA damage response." in: Genes & development, Vol. 28, Issue 18, pp. 1977-82, 2014 (PubMed).

Gauthier-Kemper, Igaev, Sündermann et al.: "Interplay between phosphorylation and palmitoylation mediates plasma membrane targeting and sorting of GAP43." in: Molecular biology of the cell, Vol. 25, Issue 21, pp. 3284-99, 2014 (PubMed).

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Catalog No. ABIN1082196
1,072.50 $
Plus shipping costs $45.00
Quantity
Price
5 x 96 tests
1,072.50 $

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