GFP-multiTrap®, 5x black 96 well plates

Application: Immunoprecipitation (IP), Mass Spectrometry (MS), Enzyme Activity Assay (EAA)

Catalog no.: ABIN1082196

Additional Information

Sample Type: Cell Extracts

Description: 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.

Characteristics: 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

Comments:

GFP-Trap® immobilized in wells
For the detection of GFP-fusion-proteins from cellular extracts.
GFP-multiTrap is available in black 96-well plate format with clear bottom for colorimetric, chemiluminescence and fluorescence detection methods.

Application Details

Reagent Preparation:
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 myg/myl
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

Assay Procedure: 1. Resuspend cell pellet (~107 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)
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 myl/well
10. Block the microtiter plate by adding 300 µl 1% milk in PBS (MPBS) to each well. Incubate for
1h at RT
11. Add 100 µl GFP-antibody (3E5, ChromoTek) at 5 myg/ml in 5% MPBS to each well and incubate 1h at RT
12. Wash the microtiter plate three times with PBS 0.05% Tween (PBST) and three times with PBS, 300 myl/well
13. Add 100 µl detection antibody (e.g. anti-rat-HRP-antibody, Jackson Immunoresearch, Cat. No. 112-035-175) at 0.4 myg/ml in 5% MPBS to each well and incubate for 1h at RT
14. Wash three times with PBST followed by three washing steps with PBS, 300 myl/well
15. Add 100 µl 3,3′,5,5′-tetramethylbenzidine solution (e.g. 1-StepTM Ultra TMB-ELISA Substrate, Thermo Fisher Pierce, Cat. No. 34028) 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 binding capacity: 1 myg GFP / well
(data obtained from HEK293T cells expressing GFP only)

Storage: Store material at 2 - 8°C, do not freeze.

Restrictions: For Research Use only

Images

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.

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.

Publications

Product: 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).