10 µg lyophilized plasmid DNA (store lyophilized plasmid at 20 C)

Transcription

1 TECHNICAL DATA SHEET BIOLUMINESCENCE RESONANCE ENERGY TRANSFER GFP 2 FUSION PROTEIN EXPRESSION VECTOR Product: Codon Humanized pgfp 2 -C Vectors Catalog number: Lot number: -001 Description: Amount: The codon humanized pgfp 2 -C vectors contain a multiple cloning site (MCS) located downstream of the Green Fluorescent Protein gene (GFP 2 ) which acts as the Acceptor moiety in a BRET 2 assay. The MCS allows for the subcloning of a gene of interest in order to create a fusion protein having the structure [GFP 2 :gene of interest]. The GFP 2 codons have been humanized to ensure higher expression levels of the fusion protein in mammalian cells. The fusion protein gene is placed under the control of the cytomegalovirus (CMV) promoter thus assuring a very high constitutive expression in a variety of cells. 10 µg lyophilized plasmid DNA (store lyophilized plasmid at 20 C) Reconstitution Protocol Reconstitution: Storage conditions: Shelf life: Centrifuge briefly to recover contents Reconstitute to 0.4 µg/µl with 25 µl of 10 mm Tris-HCl ph 8.0, 1 mm EDTA Store reconstituted plasmid at -20 C After thawing, centrifuge briefly to recover contents 1 year from date of receipt under recommended storage conditions Quality Control Procedures The identity of the codon humanized pgfp 2 -C plasmids and the presence of the MCS restriction sites are confirmed by sequence analysis. The presence of RNA and chromosomal DNA as well as the proportion of superhelical DNA are determined by agarose gel electrophoresis using 1 µg of plasmid DNA. The absence of nuclease contamination is determined by agarose gel electrophoresis following incubation of 1 µg of plasmid DNA in standard restriction buffer for 16 hours at 37 C. The quantity and purity of DNA are determined by UV spectroscopy. The functionality of the plasmids is assessed by determining GFP 2 activity upon transfection of CHO or BHK cells with LipofectAMINE. The intensity of the GFP 2 peak is compared with those obtained with reference plasmids using the FluoroCount with appropriate filters for the GFP 2. Renilla Luciferase Substrate BRET 2 requires a modified form of the Rluc coelenterazine substrate, called DeepBlueC. DeepBlueC has been selected for its ability to confer superior spectral properties to the reaction, resulting in excellent discrimination of the Rluc and GFP 2 signals.

2 Codon Humanized pgfp 2 -C1 Vector Map & Notes pgfp 2 -C1 lot number: bp SV40 early poly (A) signal TK poly (A) signal Multiple cloning site (MCS) P SV40 /P amp Zeo R f1 origin puc sequences ( ori ) Codon Humanized pgfp 2 -C1 Vector Mutiple Cloning Site Codon humanized pgfp 2 -C1 Vector TAC AAG TCC GGA CTC AGA TCT CGG CGA GCT CTC GAG AAT TCT CAC GCG BglII * SacI GFP 2 BspEI TCT GCA GGA TAT CAA GCT TGC GGT ACC GCG GGC CCG GGA TCC ACC GGA Pstl EcoRV HindIII KpnI Apal BamHl TCT AGA TAA CTG ATC A The identity of the codon humanized pgfp 2 -C1 plasmid and the presence of the MCS restriction sites have Purity (A 260 /A 280 ) at ph 8.0: 1.77 Transfection of CHO cells showed that the codon humanized pgfp 2 -C1 vector is functional and expressed

3 Codon Humanized pgfp 2 -C2 Vector Map & Notes pgfp 2 -C2 lot number: bp SV40 early poly (A) signal TK poly (A) signal Multiple cloning site (MCS) P SV40 /P amp Zeo R f1 origin puc sequences ( ori ) Codon Humanized pgfp 2 -C2 Vector Multiple Cloning Site Codon humanized pgfp 2 -C2 Vector TAC AAG TCC GGA CTC AGA TCT CCG GCG AGC TCT CGA GAA TTC TCA CGC BglII * SacI GFP 2 BspEl GTC TGC AGG ATA TCA AGC TTG CGG TAC CGC GGG CCC GGG ATC CAC CGG Pstl EcoRV HindIII KpnI Apal BamHl ATC TAG ATA ACT GAT CA The identity of the codon humanized pgfp 2 -C2 plasmid and the presence of the MCS restriction sites have Purity (A 260 /A 280 ) at ph 8.0: 1.79 Transfection of CHO cells showed that the codon humanized pgfp 2 -C2 vector is functional and expressed

4 Codon Humanized pgfp 2 -C3 Vector Map & Notes pgfp 2 -C3 lot number: bp SV40 early poly (A) signal TK poly (A) signal Multiple cloning site (MCS) P SV40 /P amp Zeo R f1 origin puc sequences ( ori ) Codon Humanized pgfp 2 -C3 Vector Multiple Cloning Site Codon humanized pgfp 2 -C3 Vector TAC AAG TCC GGA CTC AGA TCT GGC GAG CTC TCG AGA ATT CTC ACG CGT BglII * SacI GFP 2 BspEl CTG CAG GAT ATC AAG CTT GCG GTA CCG CGG GCC CGG GAT CCA CCG Pstl EcoRV HindIII KpnI Apal BamHl GAT CTA GAT A AC TGA TCA The identity of the codon humanized pgfp 2 -C3 plasmid and the presence of the MCS restriction sites have Purity (A 260 /A 280 ) at ph 8.0: 1.79 Transfection of CHO cells showed that the codon humanized pgfp 2 -C3 vector is functional and expressed

5 BRET 2 TM is a proprietary technology from BioSignal Packard. Patents pending BRET 2 TM is a trademark of Packard Instrument Company DeepBlueC is a trademark of Packard Instrument Company. Patent pending Green Fluorescent Protein (GFP 2 ) is produced and sold under license, U.S. Patent Nos. 6,020,192; 5,968,750 and 5,874,304 Cytomegalovirus (CMV) is produced and sold under license, U.S. Patent Nos. 5,1168,062 and 5,385,839 LipofectAMINE is a trademark of Life Technologies, Inc. FluoroCount is a trademark of Packard Instrument Company. LIMITED WARRANTY BioSignal Packard warrants that, at the time of shipment, the products sold by it are free from defects in material and workmanship and conform to specifications which accompany the product. BioSignal Packard makes no other warranty, express or implied, with respect to the products, including without limitation any warranty of merchantability or fitness for any particular purpose. Notification of any breach of warranty must be made within 30 days of receipt unless provided in writing by BioSignal Packard. No claim shall be honored if the customer fails to notify BioSignal Packard within the period specified. The sole and exclusive remedy of the customer for any liability of BioSignal Packard of any kind, including liability based upon warranty (express or implied whether contained herein or elsewhere), strict liability, breach of contract or otherwise, is limited, at BioSignal Packard's sole election, to replacement of the goods or a refund of the invoice price of the goods. BioSignal Packard shall not in any case be liable for special, incidental or consequential damages of any kind.