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G
reen uorescent proteins (GFP) and variants thereof are widely used to study protein localization and dynamics. However, among the most commonly used
tags for immuno-precipitation
(a brief review in Box 1), the use of GFP is limited due to the previously available anti-GFP antibod-ies, either polyclonal or monoclonal, not being comparable to those against other tags. GFP-multiTrap® is a high qual-ity 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 70°C, functional within 2 M NaCl or 0.5% SDS). From detailed in vitro binding analysis, we deter-mined that one molecule GFP-Trap® binds one molecule GFP in a stable stoichiometric complex. The dissocia-tion constant (Kd) lies with 0.59 nm within the picomolar range compara-ble to conventional antibodies. GFP-multiTrap® is available in black 96-well plate format with clear bottom for colorimetric, chemiluminescence and uorescence detection methods. With much greater stability, specicity, and afnity, GFP-Trap®, the recent addition to antibodies for immunopre-cipitation should make GFP in line to become the most suitable tags for im-munoprecipitation 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 recog-nizes 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 ex-hibit an amino acid exchange within the recognized epitope. In addition we could not detect any binding to red uorescent 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).
Chromotek-GFP-multiTrap
®
, GFP-Trap® immobilized in wells to test your GFP fusion proteins for pep-tide, protein, DNA or RNA binding.
Box 1 | Tags for Immunoprecipitation
To achieve effective immunoprecipitation, a researcher must rst overcome the difculty of nding 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 be-tween 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 pull-down. 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, specicity, and afnity, GFP-Trap®, the recent addition to antibodies for immunoprecipitation should make GFP in line to become the most suitable tags for immunoprecipitation assays.
Box 2 | Comparative Immunoprecipitation Assay
[A]
 GFP-multiTrap® Immunoprecipitations (IP) of GFP and GFP-fusion proteins (CBX1) from ex-tracts of GFP-producing human cells. Input (I), non-bound (FT), and bound (B) fractions were sepa-rated by SDS-PAGE.
[B]
Fraction input percentages of GFP and GFP-CBX1 and their GFP brightness displaying the efciency of pulldown using the GFP-multiTrap®.
 AB
 
Allele Biotech-Introducing Cost Effectiveness to Research
Protocols
1. Immunoprecipitation
•For one immunoprecipitation reaction resuspend cell pel-let (~10
7
 cells) in 200 μL 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 4°C•Transfer supernatant to a precooled tube. Adjust volume with dilution buffer to 500 μL-1000 μL. Discard pellet.The cell lysate can be frozen at this point for long-term stor-age at minus 80°C.For immunoblot analysis dilute 50 μL cell lysate with 50 μL 4x SDS-sample buffer (→refer as input)• Add 50 μL (half area GBP-Plate) or 100 μL (full area GBP plate) cell lysate per well•Incubate for 2 hours at 4°C under shaking (800rpm)For western blot analysis dilute 50 μL cell lysate with 50 μL 4x SDS-sample buffer (→refer as ow-through)•Discard remaining supernatant•Wash wells two times with 100 μL- 200 μL wash buffer • Add 100 μL dilution buffer for Fluorescence intensity mea-surements Add 10 μL 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 μL 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 purication of GFP fusion proteins with the 96-well GBP plate in half-area plates (1), the wells are blocked with 100 μL blocking buffer for 30 minutes at 4°C under shaking (800rpm)•Discard blocking solution•Equilibrated wells with 50 μL dilution buffer supplemented with 0.05% Tween• Add peptides to a nal concentration of 0.15 μM•Incubate at room temperature for 20 min under shaking (800rpm)•Discard supernatant•Wash wells two times with 100 μL wash buffer • Add 100 μL dilution buffer for Fluorescence intensity mea-surements2.2. In vitro DNA binding assay• After one-step purication of GFP fusion proteins with the 96-well GBP plate in full-area plates(1), the wells are equilibrated with 100 μL dilution buffer sup-plemented with 2mM DTT and100ng/μL BSA• Add uorescent-labeled DNA probe to a nal concentration of 0.15 μM•Incubate at room temperature for 30 min under shaking (800rpm)•Discard supernatant•Wash wells two times with 100 μL dilution buffer • Add 100 μL dilution buffer for Fluorescence intensity mea-surements2.3. In Vitro Protein-Protein binding assay• After one-step purication of GFP fusion proteins with the 96-well GBP plate in full-area plates(1), the wells are equilibrated with 100 μL dilution buffer sup-plemented with 2mM DTT and100ng/μL BSA•Prepare cell lysate of RFP-fusion protein as described for GFP-fusion proteins• Add 50 μL (half area GBP-Plate) or 100 μL (full area GBP plate) cell lysate per well•Incubate for 30 minutes at 4°C under shaking (800rpm) For western blot analysis dilute 50 μL cell lysate with 50 μL 4x SDS-sample buffer (→refer as ow-through)•Discard remainning supernatant•Wash wells two times with 100 μL- 200 μL wash buffer • Add 100 μL dilution buffer for Fluorescence intensity mea-surements. Add 10 μL 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 μL 4x SDS-sample buffer (→refer as bound)
Box 1 | Product List
GFP-multiTrap® Plates
Chromotek-GFP-multiTrap® ABP-CM-GMULT1 1 Plate ABP-CM-GMULT55 Plates
 
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Protocols
Continued
3. Fluorescence Measurements
3.1. Quantication with Tecan Innite M1000 plate reader •GFP: : 490±10 nm and 511±10 nm•RFP: 586±5 nm and 608±10 nm•TAMRA: 560±5 nm and 586±5 nm•DNA: according to labelFor quantication, uorescence intensity measurements are adjusted using standard curves from labelled probes with known concentrations.
Suggested Buffers
Lysis buffer 
20 mM Tris/HCl pH7.5150 mM NaCl0.5 mM EDTA0.5% NP-40
Optional:
1 mM PMSF has to be freshly added1x Protease Inhibitor Cocktail has to be freshly addedFor nuclear/chromatin proteins:DNase nal conc. 1 μg/μl 2.5 mM MgCl2
Dilution buffer 
20 mM Tris/HCl pH7.5150 mM NaCl0.5 mM EDTA
Wash buffer 
20 mM Tris/HCl pH7.550-500 mM NaCl0.5 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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