Chromotek-GFP-multiTrap, GFP-Trap

immobilized in wells to test your GFP fusion proteins for peptide, protein, DNA or RNA binding.

reen fluorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. However, among the most commonly used tags for immunoprecipitation (a brief review in Box 1), the use of GFP is limited due to the previously available anti-GFP antibodies, either polyclonal or monoclonal, not being comparable to those against other tags. GFP-multiTrap is a high quality GFP-bingding protein based on a single domain antibody immobilized in wells. It is characterized by a small barrel shaped structure (13 KDa, 2.5 nm X 4.5 nm) and a very high stability (stable up to 70C, functional within 2 M NaCl or 0.5% SDS). From detailed in vitro binding analysis, we determined that one molecule GFP-Trap binds one molecule GFP in a stable stoichiometric complex. The dissociation constant (Kd) lies with 0.59 nm within the picomolar range comparable to conventional antibodies. GFP-multiTrap is available in black 96-well plate format with clear bottom

Box 1 | Tags for Immunoprecipitation

To achieve effective immunoprecipitation, a researcher must first overcome the difficulty of finding usable antibodies against a target of interest. Using tags that are fused to the C- or N-terminus of the target protein is common practice. In general, while keeping mindful of the unique nuances with each biological system, choosing tags that have been tested in many situations and been proven to be non-interfering is ideal. The most commonly used tags are: FLAG, Myc, HA, V5, T7, and His, which are quite small in size and in theory less likely to interfere. GST and GFP are well documented to form self-contained and stable structures independent of their fusion partners and proven to not interfere in many cases despite their larger size (in between 20-30kD). A top choice for pulldown experiments, GST can bind to glutathione beads directly. GFP and variants are excellent tags having both the advantages of being a visualization module to follow the protein both inside cells and during pulldown. However its use is limited due to the previously available anti-GFP antibodies, either polyclonal or monoclonal, not being comparable to those against other tags. With much greater stability, specificity, and affinity, GFP-Trap, the recent addition to antibodies for immunoprecipitation should make GFP in line to become the most suitable tags for immunoprecipitation assays.

for colorimetric, chemiluminescence and fluorescence detection methods. With much greater stability, specificity, and affinity, GFP-Trap, the recent addition to antibodies for immunoprecipitation should make GFP in line to become the most suitable tags for immunoprecipitation assays. Datas from

direct comparison of the GFP-Trap with conventional antibodies will be shown in Box 2. Besides wtGFP, GFP-multiTrap can also bind to eGFP and GFPS65T as well as to YFP and eYFP. It recognizes and binds a three dimensional epitope at the beta barrel structure. Interestingly it does not bind to CFP, which is due to the fact, that CFP exhibit an amino acid exchange within the recognized epitope. In addition we could not detect any binding to red fluorescent proteins derived from DsRed (RFP-Trap is available as another product line). Meanwhile, as GFP-Trap recognizes the beta barrel structuure of GFP, it does not recognise unfolded or denatured GFP (e.g. on immunoblots).

Box 2 | Comparative Immunoprecipitation Assay A

[A] GFP-multiTrap Immunoprecipitations (IP) of GFP and GFP-fusion proteins (CBX1) from extracts of GFP-producing human cells. Input (I), non-bound (FT), and bound (B) fractions were separated by SDS-PAGE. [B] Fraction input percentages of GFP and GFP-CBX1 and their GFP brightness displaying the efficiency of pulldown using the GFP-multiTrap.

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Box 1 | Product List

GFP-multiTrap Plates
Chromotek-GFP-multiTrap ABP-CM-GMULT1 ABP-CM-GMULT5 1 Plate 5 Plates

Protocols
1. Immunoprecipitation For one immunoprecipitation reaction resuspend cell pellet (~107 cells) in 200 L lysis buffer by pipetting (or using a syringe) Place the tube on ice for 30 min with extensively pipetting every 10 min Spin cell lysate at 20.000x g for 5-10 min at 4C Transfer supernatant to a precooled tube. Adjust volume with dilution buffer to 500 L-1000 L. Discard pellet. The cell lysate can be frozen at this point for long-term storage at minus 80C. For immunoblot analysis dilute 50 L cell lysate with 50 L 4x SDS-sample buffer (refer as input) Add 50 L (half area GBP-Plate) or 100 L (full area GBP plate) cell lysate per well Incubate for 2 hours at 4C under shaking (800rpm) For western blot analysis dilute 50 L cell lysate with 50 L 4x SDS-sample buffer (refer as flow-through) Discard remaining supernatant Wash wells two times with 100 L- 200 L wash buffer Add 100 L dilution buffer for Fluorescence intensity measurements Add 10 L of elution buffer to wells and transfer it to a tube. Buffer with 1M Tris pH 7.5 to an end concentration of 100 mM Tris and dilute with 10 L 4x SDS-sample buffer (refer as bound) 2. In vitro binding assays after IP 2.1. In vitro histone-tail peptide binding assay After one-step purification of GFP fusion proteins with the 96-well GBP plate in half-area plates (1), the wells are blocked with 100 L blocking buffer for 30 minutes at 4C under shaking (800rpm) Discard blocking solution Equilibrated wells with 50 L dilution buffer supplemented with 0.05% Tween Add peptides to a final concentration of 0.15 M Incubate at room temperature for 20 min under shaking (800rpm) Discard supernatant Wash wells two times with 100 L wash buffer Allele Biotech-Introducing Cost Effectiveness to Research Add 100 L dilution buffer for Fluorescence intensity measurements 2.2. In vitro DNA binding assay After one-step purification of GFP fusion proteins with the 96-well GBP plate in full-area plates (1), the wells are equilibrated with 100 L dilution buffer supplemented with 2mM DTT and 100ng/L BSA Add fluorescent-labeled DNA probe to a final concentration of 0.15 M Incubate at room temperature for 30 min under shaking (800rpm) Discard supernatant Wash wells two times with 100 L dilution buffer Add 100 L dilution buffer for Fluorescence intensity measurements 2.3. In Vitro Protein-Protein binding assay After one-step purification of GFP fusion proteins with the 96-well GBP plate in full-area plates (1), the wells are equilibrated with 100 L dilution buffer supplemented with 2mM DTT and 100ng/L BSA Prepare cell lysate of RFP-fusion protein as described for GFP-fusion proteins Add 50 L (half area GBP-Plate) or 100 L (full area GBP plate) cell lysate per well Incubate for 30 minutes at 4C under shaking (800rpm) For western blot analysis dilute 50 L cell lysate with 50 L 4x SDS-sample buffer (refer as flow-through) Discard remainning supernatant Wash wells two times with 100 L- 200 L wash buffer Add 100 L dilution buffer for Fluorescence intensity measurements. Add 10 L of elution buffer to wells and transfer it to a tube. Buffer with 1M Tris pH 7.5 to an end concentration of 100 mM Tris and dilute with 10 L 4x SDS-sample buffer (refer as bound)

ProtocolsContinued
3. Fluorescence Measurements 3.1. Quantification with Tecan Infinite M1000 plate reader GFP: : 49010 nm and 51110 nm RFP: 5865 nm and 60810 nm TAMRA: 5605 nm and 5865 nm DNA: according to label For quantification, fluorescence intensity measurements are adjusted using standard curves from labelled probes with known concentrations. Suggested Buffers Lysis buffer 20 mM Tris/HCl pH7.5 150 mM NaCl 0.5 mM EDTA 0.5% NP-40 Optional: 1 mM PMSF has to be freshly added 1x Protease Inhibitor Cocktail has to be freshly added For nuclear/chromatin proteins: DNase final conc. 1 g/l 2.5 mM MgCl2 Dilution buffer 20 mM Tris/HCl pH7.5 150 mM NaCl 0.5 mM EDTA Wash buffer 20 mM Tris/HCl pH7.5 50-500 mM NaCl 0.5 mM EDTA Blocking Buffer 3% milk solved in TBS-T (0.075% Tween) Elution buffer (freshly prepared) 300 mM Glycin pH2.5

Box 3 | GFP-multiTrap Overview

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