GFP-multiTrap®

Details for Product No. ABIN1082196
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Target Name (Antigen)
Reactivity
Aequorea victoria
(21)
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 8 references available
Quantity 5 x 96 tests
Quantity Comment 480 reactions
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Request Want additional data for this product?

The Independent Validation Initiative strives to provide you with high quality data. Find out more

Catalog No. ABIN1082196
1,029.60 $
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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 Immunoprecipitations (IP) of GFP from protein extracts of GFP-producing human cells. Input (I), non-bound (FT) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining and Western Blotting. (hc) heavy chain, (lc) light chain of conventional antibodies.
GFP-multiTrap® (2) Comparison of GFP-Trap_A and GFP-Trap_M Left (IP): Pulldown of GFP with GFP-Trap_A and GFP-Trap_M from 293T cell extracts. Input (I) and bound (B) fractions were separated by SDS-PAGE followed by Coomassie staining.Right (Co-IP): Pulldown of GFP-PCNA with GFP-Trap_A and GFP-Trap_M from 293T cell extracts. Other bands: potential interaction partners of PCNA.
Product cited in: Wild, Farhan, McEwan et al.: "Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth." in: Science (New York, N.Y.), Vol. 333, Issue 6039, pp. 228-33, 2011 (PubMed).

Majumdar, Cesario, White-Grindley et al.: "Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory." in: Cell, Vol. 148, Issue 3, pp. 515-29, 2012 (PubMed).

Metzger, Gache, Xu et al.: "MAP and kinesin-dependent nuclear positioning is required for skeletal muscle function." in: Nature, Vol. 484, Issue 7392, pp. 120-4, 2012 (PubMed).

Gudesblat, Schneider-Pizoń, Betti et al.: "SPEECHLESS integrates brassinosteroid and stomata signalling pathways." in: Nature cell biology, Vol. 14, Issue 5, pp. 548-54, 2012 (PubMed).

Ries, Kaplan, Platonova et al.: "A simple, versatile method for GFP-based super-resolution microscopy via nanobodies." in: Nature methods, Vol. 9, Issue 6, pp. 582-4, 2012 (PubMed).

Pichler, Jack, Wolf et al.: "Versatile toolbox for high throughput biochemical and functional studies with fluorescent fusion proteins." in: PLoS ONE, Vol. 7, Issue 5, pp. e36967, 2012 (PubMed).

Castello, Fischer, Eichelbaum et al.: "Insights into RNA biology from an atlas of mammalian mRNA-binding proteins." in: Cell, Vol. 149, Issue 6, pp. 1393-406, 2012 (PubMed).

Poulsen, Madsen, Young et al.: "Using guanidine-hydrochloride for fast and efficient protein digestion and single-step affinity-purification mass spectrometry." in: Journal of proteome research, Vol. 12, Issue 2, pp. 1020-30, 2013 (PubMed).

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