A choice of application-friendly methods. LANCE Reagents and TruPoint Assay Kits

Transcription

1 A choice of application-friendly methods LANCE Reagents and TruPoint Assay Kits

2 Contents Time-resolved fluorometry LANCE time-resolved fluorescent resonance energy transfer assay....2 TruPoint time-resolved fluorescence quench assay Detection systems from assay development to uhts LANCE products and services Features and benefits LANCE kinase reagents LANCE europium labeled anti-tag antibodies LANCE Streptavidin & biotin reagents LANCE IgG detection reagents SureLight Allophycocyanin (APC) acceptor labels LANCE labeling reagents LANCE labeling service TruPoint products and services Features and benefits TruPoint Assay Kits TruPoint labeling service Recommended buffers and plates for LANCE and TruPoint assays Antibody specificities Troubleshooting LANCE and TruPoint product listing

3 Time-resolved fluorometry Time-resolved fluorometry exploits the unique fluorescence properties of lanthanide chelates. Long fluorescence decay after excitation (up to a millisecond) allows time-delayed signal detection to virtually eliminate background originating, for example, from microplates and buffer components. Large Stokes shift minimizes crosstalk, resulting in a high signal-to-background ratio. Homogeneous assays particularly benefit from time-resolved fluorometry because the sample constituents present during detection cause very high background fluorescence when conventional fluorochromes are used. There are two types of homogeneous time-resolved fluorescence assays: TruPoint assays which are based on proprietary SignalClimb technology and LANCE time-resolved fluorescence resonance energy transfer assay (TR-FRET). LANCE time-resolved fluorescent resonance energy transfer assay LANCE is based on the proximity of a donor label (europium chelate) and an acceptor label (Allophycocyanin) that have been brought together by a specific binding reaction. The excited energy of the europium chelate is transferred by a nonradiative resonance energy transfer mechanism to an acceptor within a short distance. Fluorescent lanthanide chelates with long excited state lifetimes are used to avoid interference caused by short-lived emission from acceptor molecules excited directly rather than by energy transfer. LANCE TR-FRET has been widely used, for example, in kinase, nuclear receptor and protein binding assays. TruPoint time-resolved fluorescence quench assay The homogeneous TruPoint platform utilizes the powerful, new SignalClimb technology that s based on the close proximity of two labels. One is a fluorescent lanthanide chelate and the other is a suitable organic quencher molecule. Fluorescence is strongly quenched when the labels are in close proximity, and after the separation of the labels, lanthanide fluorescence is restored. Measurement is by time-resolved fluorometry. In enzymatic cleavage assays, activity can be detected by measuring the signal increase. This proprietary technology also allows multiplexing of the assays using europium, terbium and samarium chelates. The TruPoint assay principle is well suited for cleavage assays, such as protease and helicase assays. SureLight APC as an acceptor label lyophilized form to give extended stability high lot-to-lot consistency Europium chelate as a donor label over 15 years experience industry leader patent protected 2

4 Literature LANCE & TruPoint Stability of the Wallac LANCE Eu-chelates Quench Correction for LANCE Time-Resolved Fluorescence Resonance Energy Transfer LANCE: Homogeneous Assay Platform for HTS Product information package Application note Application note Scientific article Ref # Ref # Ref # Hemmilä I. J Biomol Screening Vol 4, number 6 (1999) Detection systems from assay development to uhts Various models of EnVision, VICTOR and Fusion are available to meet your specific application needs, and your speed and capacity requirements. For ultra-fast processing of 384-well or 1536-well plates, uhts microplate imager ViewLux allows detection of all samples on a microplate simultaneously. When measuring LANCE energy transfer signal on a white 384-well microplate, all of these instruments give Z > 0.5 with reading times of approximately 1.5, 4, 13, and 14 minutes per plate on ViewLux, EnVision, VICTOR and Fusion, respectively. Based on your sensitivity requirements and throughput needs, EnVision provides flexibility to run more than 45,000 wells per day. When measuring TruPoint signal on a white 384-well plate, all of these instruments give Z > 0.5 with reading times of 1, 2.5, 8 and 9 minutes per plate on Viewlux, EnVision, VICTOR and Fusion, respectively. Based on your sensitivity requirements and throughput needs, EnVision provides flexibility to run more than 70,000 wells per day. Victor EnVision ViewLux 3

5 LANCE products and services Features Broadest range of reagents Stable reagents Stable signal Easy to miniaturize without loss of sensitivity Lot-to lot consistency Sensitive time-resolved fluorometry Benefits Suitable for many applications Allows flexibility in daily planning and budgeting Allows flexibility in scheduling daily work Economical Transfers easily from assay development to the screening laboratory Quality hits LANCE kinase reagents Protein phosphorylation is a basic mechanism for the modification of protein function in eukaryotic cells. Protein kinases use ATP to phosphorylate tyrosine, serine and/or threonine residues on their substrates. LANCE reagents can be used to detect both phosphorylated peptides and proteins in physiological ATP concentrations. Indirect serine kinase assay: Eu-labeled anti-species antibody binds to phosphoserine/threonine antibody Direct tyrosine kinase assay: Eu-labeled anti-phosphotyrosine antibody binds to phospho-peptide LANCE tyrosine kinase start-up reagents LANCE tyrosine kinase start-up reagents offer an easy method to learn the technology. In addition to tyrosine kinase reagents, the package contains the positive control data on all the PerkinElmer instrumentation with recommended instrument settings. 4

6 Type of product Amount Part no. LANCE tyrosine kinase start-up reagents 2 x 384 wells AD0121 Kit contains the following components: LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) SureLight Allophycocyanin-Streptavidin, kinase assay Positive control Substrate for the enzyme assay: Biotinylated Poly (Glu, Tyr) Buffer White 384-well OptiPlates (2 pcs) Donor labels for tyrosine kinase assays The P-Tyr-100, PT66 and PY20 antibodies are affinity purified mouse monoclonal antibodies that bind to phosphorylated tyrosine residues. The P-Tyr-100 antibody (supplied by Cell Signaling Technology, CST) is particularly effective in recognizing a wide range of peptide sequences. 10 µg is enough for 1,250 assay points, 50 µg is enough for 6,000 assay points and 1 mg is enough for 125,000 assay points when final concentration of 1 nmol/l in 50 µl volume is used (8 ng/well) Type of product Amount Part no. LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PY20) 50 µg AD0066 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PY20) 1 mg AD0067 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PT66) 50 µg AD0068 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PT66) 1 mg AD0069 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) 10 µg AD0203 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) 50 µg AD0161 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) 1 mg AD0162 Donor labels for serine/threonine kinase assays PerkinElmer has a wide variety of LANCE europium labeled serine/threonine kinase antibodies available in stock daily. PerkinElmer also offers a test service to find the specific antibody for the customer s substrate of interest. S/B ratio data is provided for substrates tested against our panel of Eu-labeled phosphoserine/threonine antibodies listed on page 6. LANCE serine/threonine kinase assays can also be designed using Eu-labeled anti-species antibodies against specific anti-phospho antibodies (application note # ). 10 µg is enough for 1,250 assay points, 50 µg is enough for 6,000 assay points and 1 mg is enough for 125,000 assay points when final concentration of 1 nmol/l in 50 µl volume is used (8 ng/well). 5

7 Type of product Amount Part no. LANCE Eu-W1024 labeled anti-phospho-threonine antibody (CST cat #9381) 10 µg AD0094 LANCE Eu-W1024 labeled anti-phospho-threonine antibody (CST cat #9381) 1 mg AD0095 LANCE Eu-W1024 labeled anti-phospho-threonineproline antibody (CST cat #9391) 10 µg AD0099 LANCE Eu-W1024 labeled anti-phospho-threonineproline antibody (CST cat #9391) 1 mg AD0100 LANCE Eu-W1024 labeled anti-phospho-serine/threonine antibody (Upstate Biotechnology cat #44-006) 10 µg AD0176 LANCE Eu-W1024 labeled anti-phospho-serine/threonine phenylalanine antibody (CST cat #9631) 10 µg AD0178 LANCE Eu-W1024 labeled anti-phospho-serine/threonine proline antibody (Upstate Biotechnology cat #05-368) 10 µg AD0180 LANCE Eu-W1024 labeled anti-phospho-(thr) PKA substrate antibody (CST cat #9621) 10 µg AD0182 LANCE Eu-W1024 labeled anti-phospho-(thr) Akt substrate antibody (CST cat #9611) 10 µg AD0184 LANCE Eu-W1024 labeled anti-phospho-serine antibody (Zymed cat # ) 10 µg AD0186 LANCE Eu-W1024 labeled anti-phospho-(ser) PKC substrate antibody (CST cat #2261) 10 µg AD0188 LANCE Eu-W1024 labeled anti-phospho-(ser) binding motif (CST cat #9601) 10 µg AD0190 Acceptor labels for kinase assays Allophycocyanin-Streptavidin, kinase assay product, has been designed particularly for kinase applications by optimizing the APC and Streptavidin ratio. It is provided in lyophilized form to provide maximum stability and lot-to-lot consistency. 1 mg is enough for 6,000 assay points and 50 mg is enough for 250,000 assay points when final concentration of 25 nmol/l in 50 µl volume is used (200 ng/well). Type of product Amount Part no. SureLight Allophycocyanin-Streptavidin, kinase assay 1 mg CR SureLight Allophycocyanin-Streptavidin, kinase assay 50 mg CR

8 Literature How to optimize LANCE tyrosine kinase assay Measurement of p38/mapk activity using LANCE Surelight Allophycocyanine bring new light to your HTS Comparison of Streptavidin: SureLight Allophycocyanin to other Manufacturer's Allophycocyanin Products Model report: Eu-labeled anti-phosphoserine/ threonine antibody specificity test service Detection of insulin receptor tyrosine kinase activity using time-resolved fluorescence energy transfer technology cgmp-dependent Protein Kinase Assay for High Throughput Screening Based on Time-Resolved Fluorescence Resonance Energy Transfer Effect of assay technology and ATP concentration on IC50 values of protein kinase inhibitors Drueckes P., Totzke F., Schumacher R., Marme D. and Schächtele C. Miniaturization of a LANCE assay Ollikka P., Hemmilä I., Kivelä P., Väisälä M. and Blomberg K. Homogeneous Time-Resolved Fluorometric Energy Transfer Assay (LANCE) for Protein Tyrosine Kinase Hemmilä I. and Ahola T. Performance of Corning s 2 ul 1536 Microplate in Miniaturized TR-FRET Tyrosine Kinase and Fluorescence Polarisation Assays Pardo A. M., Martin E. M. and Vaughan L. M. Corning White 10 ul 1536 Assay Plate Performance in a Miniaturized TR-FRET Tyrosine Kinase Assay Martin E. M., Pardo A. M., Tanner A. J. and Vaughan L. M. An automated Simultaneous Screen for the Identification of Inhibitors of the Catalytic Kinase Domains of two Unrelated Receptor Tyrosine Kinase Zuck P., Cassaday J., Peltier R., Strulovici B., Iglese Jim Application note Application note Flyer Technical Bulletin Technical note Scientific article Scientific article Poster Paper presented at the 7th Annual Conference and Exhibition of the Society for Biomolecular Screening, Baltimore, Sept 2001 Poster Paper presented at the 4th Annual Conference and Exhibition of the Society for Biomolecular Screening, Baltimore, Sept 1998 Poster Paper presented at the 3rd Annual Conference and Exhibition of the Society for Biomolecular Screening, California, Sept 1997 Poster Paper presented at the 7th Annual Conference and Exhibition of the Society for Biomolecular Screening, Baltimore, Sept 2001 Poster Paper presented at the 7th Annual Conference and Exhibition of the Society for Biomolecular Screening, Baltimore, Sept 2001 Poster Paper presented at the 7th Annual Conference and Exhibition of the Society for Biomolecular Screening, Baltimore, Sept 2001 Ref # Ref # Ref # Ref # Biazzo-Ashnault et al. (2001) Anal. Biochem. 291, Bader, B. et al. (2001) J. Biomol. Screening 6, To download pdf file: To download pdf file: To download pdf file: To download pdf file: To download pdf file: To download pdf file: 7

9 LANCE europium labeled anti-tag antibodies With LANCE Eu-W1024 labeled anti-tag antibodies and SureLight APC acceptor labels (see p. 10) it is easy to set up different types of assays like protein binding, transferase and nuclear receptor assays. LANCE transferase assay LANCE nuclear receptor assay In an assay, proteins (e.g. one biotinylated, the other a GST fusion) are allowed to interact in the presence of library compounds in microtitration plates. The assay buffer should be optimized to favor interaction between proteins and to prevent binding of the assay components onto the plate. After incubating for a required time, detection reagents (e.g. LANCE Eu-labeled anti-gst and Streptavidin-APC) are added. If the final concentration of biotinylated protein is 50 nm, then the suitable final concentration for Streptavidin-APC is nm. If the final concentration of the GST fusion protein is 10 nm, then Eu-labeled anti-gst could be used at a concentration between 1 and 5 nm. Type of product Amount Part no. LANCE Eu-W1024 labeled anti-gst antibody 10 µg AD0252 LANCE Eu-W1024 labeled anti-gst antibody 50 µg AD0253 LANCE Eu-W1024 labeled anti-gst antibody 1 mg AD0254 LANCE Eu-W1024 labeled anti-ha antibody 50 µg AD0084 LANCE Eu-W1024 labeled anti-ha antibody 1 mg AD0085 LANCE Eu-W1024 labeled anti-6xhis antibody 10 µg AD0205 LANCE Eu-W1024 labeled anti-6xhis antibody 50 µg AD0110 LANCE Eu-W1024 labeled anti-6xhis antibody 1 mg AD0111 LANCE Eu-W1024 labeled anti-c-myc antibody 10 µg AD0206 LANCE Eu-W1024 labeled anti-c-myc antibody 50 µg AD0114 LANCE Eu-W1024 labeled anti-c-myc antibody 1 mg AD0115 8

10 Literature Development of a Ubiquitin Transfer Assay for High Throughput Screening by Fluorescence Resonance Energy Transfer Identification of a Nuclear Receptor for Bile Acids A Natural Product That Lowers Cholesterol As an Antagonist Ligand for FXR Development of a Homogeneous, Fluorescence Resonance Energy Transfer-Based in vitro Recruitment Assay for Peroxisome Proliferator- Activated Receptor δ via Selection of Active LXXLL Coactivator Peptides Scientific article Scientific article Scientific article Scientific article Boisclair, M.D. et al. (2000) J. Biomol. Screening 5, Makishima, M. et al. (1999) Science 284, Urizar, N.L. et al. (2002) Science 296, Drake, K.A. et al. (2002) Anal. Biochem. 304, LANCE streptavidin and biotin reagents Streptavidin labeled with Eu-W1024 chelate is suitable for most of the biotin binding applications since it is stable in neutral assay buffer ph 6-8. However, if there is lower ph or high chelating agent concentrations (>20 mm EDTA) another LANCE chelate is recommended: Eu-W8044. It is stable at ph 4-8 and stands EDTA up to 250 mm concentration at neutral ph. For more information see the Application Note Eu-W1024-biotin can be used as control for compound quenching interference studies together with Streptavidin-APC, for example, in the secondary screening. Type of product Amount Part no. LANCE Eu-W8044 labeled Streptavidin 50 µg AD0060 LANCE Eu-W8044 labeled Streptavidin 1 mg AD0061 LANCE Eu-W1024 labeled Streptavidin 50 µg AD0062 LANCE Eu-W1024 labeled Streptavidin 1 mg AD0063 LANCE Eu-W1024-biotin 10 nmol CR Literature A High-Throughput Homogeneous Assay for Reverse Transcriptase Using Generic Reagents and Time-Resolved Fluorescence Detection Bile Acids: Natural Ligands for an Orphan Nuclear Receptor Scientific article Scientific article Zhang, J.-H., et al. (2000) Anal. Biochem. 281, Parks, D.J. et al. (1999) Science 284,

11 LANCE IgG detection reagents LANCE IgG detection reagents are mainly used to set up an indirect assay to detect primary antibody, such as phosphoserine/threonine antibody (see page 4). By using these off-the-shelf reagents there is no need to label the assay specific antibody, allowing quick assay development. Type of product Amount Part no. LANCE Eu-W1024 labeled anti-human antibody 10 µg AD0212 LANCE Eu-W1024 labeled anti-human antibody 50 µg AD0074 LANCE Eu-W1024 labeled anti-human antibody 1 mg AD0075 LANCE Eu-W1024 labeled Protein G 10 µg AD0211 LANCE Eu-W1024 labeled Protein G 50 µg AD0070 LANCE Eu-W1024 labeled Protein G 1 mg AD0071 LANCE Eu-W1024 labeled anti-mouse antibody 50 µg AD0076 LANCE Eu-W1024 labeled anti-mouse antibody 1 mg AD0077 LANCE Eu-W1024 labeled anti-rabbit antibody 50 µg AD0082 LANCE Eu-W1024 labeled anti-rabbit antibody 1 mg AD0083 SureLight Allophycocyanin (APC) acceptor labels Allophycocyanin (APC) is a fluorescent, light harvesting protein unique to cyanobacteria and red algae, and it s a member of the phycophiliprotein family of direct fluorescent dyes. It has become one of the best choices of acceptors for LANCE assays used in high throughput screening. All SureLight products are provided in lyophilized form to improve long term stability and to provide easy handling of the product. In addition, SureLight products have shown a high lot-to-lot consistency over the years. 1 mg is enough for 6,000 assay points and 50 mg is enough for 250,000 assay points when final concentration of 25 nmol/l in 50 µl volume is used (200 ng/well). SureLight Allophycocyanin-Streptavidin, kinase assay, has a higher APC ratio to Streptavidin than the SureLight Allophycocyanin-Streptavidin designed for protein binding assays. Lower APC ratio is better suited for assays where the steric hindrance might be an issue. Type of product Amount Part.No SureLight Allophycocyanin-Streptavidin, kinase assay 1 mg CR SureLight Allophycocyanin-Streptavidin, kinase assay 50 mg CR SureLight Allophycocyanin-Streptavidin, protein binding assay 1 mg AD0201 SureLight Allophycocyanin-Streptavidin, protein binding assay 30 mg AD0202 SureLight Allophycocyanin-anti-c-myc antibody 1 mg AD0059C SureLight Allophycocyanin-anti-FLAG antibody 1 mg AD0059F SureLight Allophycocyanin-anti-GST antibody 1 mg AD0059G 10

12 Type of product (cont d) Amount Part no. SureLight Allophycocyanin-anti-mouse antibody 1 mg AD0059M SureLight Allophycocyanin-anti-rabbit antibody 1 mg AD0059R SureLight Allophycocyanin-anti-6xhis antibody 1 mg AD0059H SureLight Allophycocyanin-WGA 1 mg AD0059W Literature SureLight Allophycocyanin brings new light to your HTS Comparison of Streptavidin: SureLight Allophycocyanin to other Manufacturer's Allophycocyanin Products Flyer Technical Bulletin Ref # LANCE labeling reagents LANCE labeling reagents allow you to label your own biomolecule, most commonly via free amino groups (on N-terminus or lysine residues) or via SH-groups on cysteines. Detailed instructions are provided with the labeling reagent, along with quality control instructions to measure the lanthanide chelate ratio per biomolecule. LANCE chelates do not have an inner filter effect, allowing high labeling ratios. LANCE chelates have also been optimized to allow gentle labeling conditions to retain the biological activity of the biomolecule. Normally the labeled compound is as stable as the intact biomolecule and can be stored either at + 4 C or at 20 C. Wallac brand lanthanide chelates can be divided into two categories: non-fluorescent and fluorescent. Non-fluorescent chelates are used in DELFIA assays. Fluorescent chelates are used in homogeneous LANCE assays without a separation step or additional enhancement step. Chelates are available with the following reactive groups for labeling purposes: Isothiocyanate (ITC) activated chelates react primarily with free amine groups such as the ones at the N-terminus of protein or lysine residues. Iodoacetamido (IAA) activated chelates react with sulfhydryl groups on peptides and proteins forming stable covalent thioether bonds. Amino chelates react with carboxyl groups on the compound to be labeled in the presence of carbodiimides. Dichlorotriazine (DTA) activated chelates are suitable for labeling proteins, peptides, nucleic acids and small molecules containing amino, hydroxy and mercapto groups. 100 µg vials are supplied in lyophilized form and provide enough chelates for labeling about 1 mg of protein. 1 mg vials are supplied in powder form and provide enough chelates for labeling about 10 mg of protein. Yield after labeling and purification is typically 70-80%, but can vary depending on the amount and concentration of a protein. Sometimes pre-purification of a protein is needed which decreases the labeling yield. Type of product Amount Part no. LANCE Eu-W1024 ITC chelate & Eu standard 100 µg AD0096 LANCE Eu-W1024 ITC chelate & Eu standard 1 mg AD0013 LANCE Eu-W1284 iodoacetamido chelate & Eu standard 100 µg AD0107 LANCE Eu-W1284 iodoacetamido & Eu standard 1 mg AD0014 Other activations for these chelates are available upon request. Please contact labellingservices@perkinelmer.com 11

13 For assays that require a high EDTA concentration, a high temperature or a ph below 7, the W8044 series of Eu-chelates is a better choice because of their greater stability. Type of product Amount Part no. LANCE Eu-W8044 DTA chelate & Eu standard 1 mg AD0020 Other activations for W8044 chelate are available upon request. Please contact labellingservices@perkinelmer.com Literature A PDZ Domain-Based Detection System for Enzymatic Assays Protein Protein Interactions: Development of FRET Assay Measuring CD40L and 5C8 Interactions Jarpe M., Glover G. and Josiah S. Scientific article Poster Ferrer, M. et al. (2002) Anal. Biochem. 301, To download pdf file: LANCE labeling service LANCE labeling service is an excellent alternative for labeling your biomolecules when you don t have the time or the manufacturing capabilities to label it yourself. PerkinElmer offers you our expertise and modern equipment and facilities in protein, peptide and oligonucleotide labeling and purification. Custom peptide synthesis is performed by an automated peptide synthesizer using Fmoc chemistry. After synthesis, the peptide will be analyzed using reversed phase HPLC and mass spectrometry. Labeled LANCE and TruPoint peptides are purified with reversed phase HPLC. Here, a PerkinElmer instrument with a Diode Array Detector (PerkinElmer 200 Series) is used for a TruPoint peptide purification. The labeled TruPoint peptide contains both a fluorescent Eu-label and a quencher molecule. 12

14 Protein labeling (MW > 5000 Da) PerkinElmer has the capability to label custom proteins from 100 µg to 50 mg in one batch. Proteins should be provided without carrier proteins (such as BSA or gelatine) in the buffer. If the buffer contains azide, Tris or other free amines, prepurification is needed and the yield will be lower. Purification of labeled protein is done with gel filtration. Labeled proteins are supplied in Tris based buffer with 0.1% BSA and shipped at + 4 C, if not otherwise instructed. The lanthanide chelate ratio to protein is provided with protein concentration information in the Product Information Sheet along with the labeling date. Lanthanide chelates do not normally affect the stability of the biomolecule, thus the long term storage of the labeled protein should follow the requirements of the biomolecule either at + 4 C or at -20 C. To get a quotation for custom protein labeling, please contact labellingservices@perkinelmer.com. Peptide labeling (< 40 amino acids or MW < 5000 Da) PerkinElmer s Labeling Service department offers custom labeling of peptides. With our automated peptide synthesizer, we are able to produce peptide sequences with lanthanide labeling based on the customer s requirements. It is also possible for customers to ship their peptides to us for lanthanide labeling. For quotations contact labellingservices@perkinelmer.com Labeling during synthesis Labeling post synthesis Labeling site Synthetic building block can be added to any place of the sequence Lysines or cysteines N-terminus Labeling ratio Can be defined by the customer Depends on the number of Lys/Cys in the peptide 1 Note Patented technology No lysines in the peptide Oligonucleotide labeling (maximum length of 55 base pairs) Custom oligonucleotides contain the bases specified by the customer and the modified bases with free amino groups to be labeled after synthesis. The quotation contains the synthesis, labeling and purification of the oligonucleotide. Labeled oligonucleotide is normally purified by UREA-PAGE or by HPLC when needed. For quotations please contact labellingservices@perkinelmer.com 13

15 TruPoint Assay Kits and labeling service Features Measures increase in signal Time-resolved fluorescence Emission at 615 nm, red-shifted High quenching efficiency, high S/B ratios Stable signal Stable reagents Readily miniturizable to 1536 well assay Multiplexing capability Benefits Sensitive, lower false positive rate More sensitive, less enzyme needed, enables kinetic measurements Higher quality hits Robust screening Results can be read over several days Allows flexibility to screening schedules Affordable Provides more information at lower cost TruPoint Caspase Assay Kits The unique combination of time-resolved fluorometry and signal increase measurement provides extra sensitive caspase assays. Proprietary SignalClimb technology provides high quenching efficiency and purity of quenched peptides, allowing the measurement of high Z ratios (even with low enzyme activities). Type of product Amount Part no. TruPoint Caspase-3 Assay Kit 2 x 384 wells AD0125 TruPoint Caspase-3 Assay Kit 50 x 384 wells AD0126 TruPoint Caspase-6 Assay Kit 2 x 384 wells AD0146 TruPoint Caspase-6 Assay Kit 50 x 384 wells AD0147 TruPoint Caspase-8 Assay Kit 2 x 384 wells AD0148 TruPoint Caspase-8 Assay Kit 50 x 384 wells AD0149 TruPoint Caspase-3 Assay Kit 96 3 x 96 wells AD0245 TruPoint Caspase-3 Assay Kit 96 w/o plates 288 assays AD0246 Kit contains the following components: Caspase reaction buffer DTT (dithiothreitol) Caspase substrate Black 384-well or 96-well OptiPlates 14

16 TruPoint Caspase reagents sold separately: Type of product Amount Part no. TruPoint Caspase Reaction Buffer 1 L CR TruPoint Caspase-3 Substrate, 5x90 nmol 96,000 assays CR TruPoint Caspase-6 Substrate, 5x90 nmol 96,000 assays CR TruPoint Caspase-8 Substrate, 5x90 nmol 96,000 assays CR Lysis Buffer 30 ml OptiPlate-384 F 50 plates Caspase Reaction Buffer contains: Pipes, ph 7.4 NaCl, EDTA, CHAPS Sucrose NaN 3 (DTT is not included in the buffer) Literature TruPoint Caspase-3 assay with one step cell handling Homogeneous TruPoint assay for Caspase-6 or Caspase-8 detection Multiparametric Homogeneous Time-Resolved Fluorescence Quenching (TruPoint ) Assay for Detection of Proteases Homogeneous Time-Resolved Fluorescence Quenching Assay for Caspase-3 Sensitive Homogeneous Protease Assay Based on Time-resolved Fluorometry Karvinen J., Andersson K., Hurskainen P., Hovinen J. and Hemmilä I. Application note Application note Poster handout Scientific article Poster Paper presented at the 6 th Annual Conference and Exhibition of the Society for Biomolecular Screening, Vancouver, Sept 2000 Ref # Ref # Ref # Karvinen, J. et al. (2002) J. Biomol. Screening 7, To download pdf file: TruPoint Helicase Assay Kit The TruPoint helicase kit is able to measure the increase in signal at several intervals, allowing the kinetics of the assay to be determined. The signal is stable overnight. A double-stranded DNA substrate for a helicase assay has two complementary strands (one labeled at 5 end with Eu chelate and the other labeled at 3 end with a quencher). The substrate is supplied in precipitate. 15

17 Type of product Amount Part no. TruPoint Helicase Assay Kit 4x384 wells AD0165 Kit contains the following components: Helicase Substrate Capture Strand White 384 OptiPlate Components sold separately: Type of product Amount Part no. TruPoint Helicase Capture Strand, 15 nmol 19,200 assays AD0164 TruPoint Helicase Substrate, 5x0.6 nmols 19,200 assays AD0166 Literature Time-Resolved Fluorescence Energy Transfer DNA Helicase Assays for High Throughput Screening. Scientific article Earnshaw, D.L. et al. (1999) J. Biomol. Screening 4, TruPoint peptide labeling service PerkinElmer has developed a labeling and purification process that allows TruPoint peptides to be delivered with high purity and high quenching efficiency. Normally, the amino end of the peptide is labeled with LANCE chelate and C-terminus with a quencher dye, but the quencher or the LANCE chelate can be added in the middle of the peptide as well. The distance between labels in the solution is the key issue on the final quench percent. For short peptides (< 6 aa) normally > 99% quench is reached. For longer peptides (8-12 aa) normally > 95% quench is reached. Why high quenching efficiency is needed? 99.9% quenched substrate max S/B = % quenched substrate max S/B = % quenched substrate max S/B = % quenched substrate max S/B = 9 Literature A Homogeneous 384-Well High Throughput Screen for Novel Tumor Necrosis Factor Receptor: Ligand Interactions Using Time-Resolved Energy Transfer. Scientific article Moore, K.J. et al. (1999) J. Biomol. Screening 4,

18 Recommended buffers and plates for LANCE and TruPoint assays In addition to offering many assay technologies for high throughput screening, PerkinElmer has a wide selection of microtiter plates, detection devices, disposables and reagents. Since we are a complete solutions provider, we are able to offer guidelines that will optimize any high throughput screening assay that you are performing, saving you time during the assay development phase. A wide variety of plates can be used with LANCE and TruPoint technologies Plate selection is dependent upon the desired microtiter plate density, assay technology and detection instrument to be used. LANCE and TruPoint assays can be run in any plate density and color: 96, 384 or 1536 well plates, clear, black or white. However, slightly different results are obtained when different detection devices are used. Thus the following plate selection recommendatations for LANCE assays are given: Optimize the buffer by knowing the application and technology needs The best buffer to be used with LANCE Eu-W1024 chelate is TRIS based. The addition of % of BSA is recommended to prevent the labeled molecules from adsorbing onto a microtiter well wall during the incubation step % BSA is recommended when higher plate densities and automated liquid handling is used. Higher concentrations of BSA might form air bubbles which affect the presicion of the assay. Detection instrument EnVision VICTOR ViewLux Fusion Recommended plates White or black 384-well OptiPlates White 384-well OptiPlates Normally, there is no need to add detergent to LANCE assays, but Tween40 or Tween20 is recommended at a concentration between 0.01% and 0.1% when very sticky compounds are used. Additionally, Chaps ( %), Brij35 ( %) and Triton X-100 can be used as a detergent. With EnVision, VICTOR and ViewLux, both white and black 384-well OptiPlates can be used, having little effect on detection limit or z. With Fusion, white 384-well OptiPlates provide better sensitivity on LANCE assays than black plates. Phosphate buffer is not recommended to be used with Eu-W1024 chelate. When no other buffer can be used, we recommend changing the chelate to Eu-W8044. Also, when >20 mm EDTA concentration, Mn 2+ or ph <6 are used, Eu-W8044 is recommended. For more details please see the Application note #

19 Preservatives such as NaN 3 can be used in the buffer without any effect on assay results. Adding 0.1% of the Stabilizer to the TRIS based buffer is recommended when storing Eu-labeled compounds for long periods. Use of the Stabilizer in the final assay buffer is also recommended to prevent the biomolecules from adsorbing to the plastics during the assay incubation. Stabilizer contains 7.5% BSA in Tris buffer. BSA is carefully selected and highly purified to remove any trace amounts of heavy metals. For TruPoint assays, the same rules apply. The buffer is dictated by the assay. Most often it is either Tris-HCl or Hepes. If the enzyme requires a heavy metal ion (such as Mn(II), Zn(II)) for activity, then reaction has to be stopped when the final EDTA concentration is times the concentration of the divalent cation. Type of product Amount Part no. LANCE detection buffer 10x 250 ml CR Stabilizer (7.5% BSA) 50 ml CR White 384-well OptiPlates 50 plts LANCE detection buffer is a 10-fold concentrate of Tris-HCL buffer containing purified BSA, ph 7.5 Antibody specificities Anti-Phosphothreonine/serine antibodies Cat. number Antibody type Antibody source Epitope Peptide substrates* AD0094 AD0095 polyclonal rabbit IgG CST cat# 9381 T* VTKT*PP: 4E-BP1 (Thr70) AD0099 AD0100 monoclonal mouse IgM CST cat# 9391 X-T*-P-X QTPT*PTR: ATF-2(Thr71) AD0176 monoclonal mouse IgG Upstate Biotechnology cat# Proprietary mixture of two mouse monoclonal antibodies that recognize a broad subset of serine/threonine phosphorylated proteins AD0178 polyclonal rabbit IgG CST cat# 9631 Y/F/W-S*/T*-F RGFS*FVA: p90rsk (Ser380) AD0182 polyclonal rabbit IgG CST cat# 9621 R-X-X-T*-X-X and/or R-R-X-S*-X-X RRPS*YRK: CREB (Ser133) AD0184 polyclonal rabbit IgG CST cat# 9611 R/K-X-R/K-X-X-S*/T*-X-X RTWT*LCG: PKA (Thr197) AD0186 polyclonal rabbit IgG Zymed No significant cross-reactivity to phosphorylated threonines or tyrosines AD0188 polyclonal rabbit IgG CST cat# 2261 X-X-R/K-X-S*-Hyd-R/K RKRS*RKE: Histone H2B (Ser32) AD0190 polyclonal rabbit IgG CST cat# 9601 X-R/K-X-X-S*-X-P RSPS*MPE: cdc25 (Ser216) * These are optimal peptide substrates. Each of these motif specific antibodies may recognize hundreds of phosphopeptide antigens. Please inquire with Cell Signaling Technology for other substrate sequences (bulks@cellsignal.com). 18

20 IgG detection Anti-tag antibodies Anti-mouse antibody AD0076, AD0077 Type: rabbit polyclonal antibody Specificity: reacts with all mouse IgG subclasses. The reaction with IgG1, IgG2a and IgG2b is somewhat stronger than the reaction with IgG3. The antibody also reacts with mouse IgA and IgM. Cross reaction with human immunoglobulins is less than 0.2% with fetal calf serum less than 0.1% and with rat serum and rat IgG less than 3%. Cross-reactions with goat, guinea pig, ox and swine immunoglobulins is less than 1.5% when determined with ELISA. AD0059M Type: goat polyclonal antibody Specificity: cross-reactivity to other species has not been tested. Anti-rabbit antibody AD0082, AD0083 Type: goat polyclonal antibody Specificity: reacts with all classes of rabbit immunoglobulins. Cross-reaction with human and mouse immunoglobulins is less than 0.7%, with ox, rat and swine immunoglobulins and fetal calf serum less than 0.1% and with guinea pig immunoglobulins about 20% when determined with ELISA. AD0059R Type: goat polyclonal antibody Specificity: cross-reactivity to other species has not been tested. Anti-HA antibody Type: mouse monoclonal antibody, IgG2b subclass Specificity: epitope sequence YPYDVPDYA derived from human influenza hemagglutinin (HA) protein. Anti-c-myc antibody Type: mouse monoclonal antibody, IgG1 subclass Specificity: epitope sequence EQKLISEEDL derived from the human c-myc protein. Anti-GST antibody AD0252, AD0253, AD0254 Type: goat polyclonal antibody AD0059G Type: mouse monoclonal antibody Anti-6xHis antibody: Type: mouse monoclonal antibody, IgG1 subclass Specificity: recognizes polyhistidine tags localized at the amino- or carboxyl-terminus. Anti-FLAG (M2) antibody Type: mouse monoclonal antibody, IgG1 subclass Specificity: recognizes FLAG sequence DYKDDDK at N-terminus, Met-N-terminus or c-terminus of FLAG fusion proteins. Anti-human antibody Type: mouse monoclonal antibody, IgG2b subclass Specificity: specific for the Fc part of human IgG. The antibody recognizes all IgG subclasses and cross-reacts with rabbit immunoglobulins. Protein G Type: recombinant Protein G cloned from Streptococcus spp. and produced in E.coli. Specificity: binds to the Fc region of IgG by a non-immune mechanism, which is similar to that of Protein A, but has a broader range and higher binding affinity. It binds to all subclasses of human and mouse IgG. In addition, it binds to rat, goat, sheep, guinea pig, rabbit cow, pig and horse antibodies. It does not bind to chicken or cat antibodies, and binds weakly to IgG from dog. It does not bind to human IgA, IgM or serum albumin. 19

21 Troubleshooting of LANCE and TruPoint assays Problem Too low signal or S/B ratio Decrease in assay counts after storage of labeled components or due to the interfering substances in assays. Poor reproducibility of samples: How to improve precision Possible cause/effect High background Low specific signal Eu-labeled component not properly stored Heavy metal contamination of BSA Heavy metal ion in buffer (like Mn 2+ and Zn 2+ ) quenches Eu fluorescence and energy transfer signal High concentrations of chelators in assays Low ph of assay buffer Sticky reagents Color or chemical quenching of the library compounds Problems with pipetting or dispensing Solution Decrease TruPoint substrate concentration/ decrease concentration of Eu-labeled component Labeled compound has some free chelate; repurify the label Purity of the biomolecule labeled with the chelate has not been high enough (at least 50% purity) Make sure that LANCE reagents are used Do not use a colored assay buffer Increase concentration of Eu-component In a binding reaction increase concentration of acceptor label Biotinylated reagent in the assay: make sure that reagent is really biotinylated and all unreacted biotin has been removed Do not store APC-labeled components frozen Direct assay gives higher signal than indirect assay in LANCE assays Increase enzyme concentration Lyophilized TruPoint substrates can be stored at +4 C but after reconstitution keep at -20 C or colder Do not store labeled components diluted (concentration less than 1 mm) Store Eu-labeled components in Tris-HCl buffer, ph Add purified stabilizing agents like glycerol, glucose, BSA (product # CR84 100) Heavy metal ions can be chelated by adding EDTA times the concentration of the divalent cation prior to the measurement High concentrations of chelators (>0.1 mm) should be avoided in the absence of a divalent cation when working with Eu-W1024-labeled components, but Eu-W8044 chelate withstands up to 10 mm EDTA at neutral ph in the absence of a divalent cation When working with Eu-W1024-labeled components the ph must not be below 7, but Eu-W8044 chelate withstands ph down to 5 Use polypropylen tubes in storage and dilutions Use a proper detergent or % BSA to prevent adhering onto tubes and wells Use quench correction software for LANCE assay Decrease the total volume of the assay: in 96-well plates 50 µl and in 384-well plates µl Black plates decrease quenching problems Make sure there is no carryover Use Eu signals in TR-FRET to evaluate pipetting/dispensing accuracy 20

22 LANCE and TruPoint product listing Product code Product Package AD0013 LANCE Eu-W1024-ITC chelate & Eu std 1 mg AD0014 LANCE Eu-W1284 iodoacetamido chelate & Eu std 1 mg AD0020 LANCE Eu-W8044 DTA chelate & Eu std 1 mg AD0059C SureLight Allophycocyanin-anti-c-Myc 1 mg AD0059F SureLight Allophycocyanin-anti-FLAG 1 mg AD0059G SureLight Allophycocyanin-anti-GST 1 mg AD0059H SureLight Allophycocyanin-anti-6His 1 mg AD0059M SureLight Allophycocyanin-anti-Mouse 1 mg AD0059R SureLight Allophycocyanin-anti-Rabbit 1 mg AD0059W SureLight Allophycocyanin-wheat germ agglutinin 1 mg AD0060 LANCE Eu-W8044 labeled streptavidin 50 µg AD0061 LANCE Eu-W8044 labeled streptavidin 1 mg AD0062 LANCE Eu-W1024 labeled streptavidin 50 µg AD0063 LANCE Eu-W1024 labeled streptavidin 1 mg AD0066 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PY20) 50 µg AD0067 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PY20) 1 mg AD0068 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PT66) 50 µg AD0069 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (PT66) 1 mg AD0070 LANCE Eu-W1024 labeled Protein G 50 µg AD0071 LANCE Eu-W1024 labeled Protein G 1 mg AD0074 LANCE Eu-W1024 labeled anti-human IgG 50 µg AD0075 LANCE Eu-W1024 labeled anti-human IgG 1 mg AD0076 LANCE Eu-W1024 labeled anti-mouse IgG 50 µg AD0077 LANCE Eu-W1024 labeled anti-mouse IgG 1 mg AD0082 LANCE Eu-W1024 labeled anti-rabbit IgG 50 µg AD0083 LANCE Eu-W1024 labeled anti-rabbit IgG 1 mg AD0084 LANCE Eu-W1024 labeled anti-ha antibody 50 µg AD0085 LANCE Eu-W1024 labeled anti-ha antibody 1 mg AD0094 LANCE Eu-W1024 labeled anti-phosphothreonine antibody (CST) 10 µg AD0095 LANCE Eu-W1024 labeled anti-phosphothreonine antibody (CST) 1 mg AD0096 LANCE Eu-W1024-ITC chelate & Eu std 100 µg AD0099 LANCE Eu-W1024 labeled anti-phosphothreonine-proline antibody (CST) 10 µg AD0100 LANCE Eu-W1024 labeled anti-phosphothreonine-proline antibody (CST) 1 mg AD0107 LANCE Eu-W1284 iodoacetamido chelate & Eu std 100 µg AD0110 LANCE Eu-W1024 labeled anti-6xhis antibody 50 µg AD0111 LANCE Eu-W1024 labeled anti-6xhis antibody 1 mg AD0114 LANCE Eu-W1024 labeled anti-c-myc antibody 50 µg AD0115 LANCE Eu-W1024 labeled anti-c-myc antibody 1 mg AD0121 LANCE Tyrosine Kinase Start up reagents 2 plates AD0125 TruPoint Caspase-3 Assay Kit 2 plates AD0126 TruPoint Caspase-3 Assay Kit 50 plates AD0146 TruPoint Caspase-6 Assay Kit 2 plates AD0147 TruPoint Caspase-6 Assay Kit 50 plates AD0148 TruPoint Caspase-8 Assay Kit 2 plates 21

23 LANCE and TruPoint product listing (cont d) Product code Product Package AD0149 TruPoint Caspase-8 Assay Kit 50 plates AD0161 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) 50 µg AD0162 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) 1 mg AD0164 TruPoint Helicase Capture strand points AD0165 TruPoint Helicase Assay Kit 4 plates AD0166 TruPoint Helicase Substrate points AD0176 LANCE Eu-W1024 labeled phospho-serine/threonine antibody (Upstate) 10 ug AD0178 LANCE Eu-W1024 labeled phosphoserine/threonine phenylalanine antibody (CST) 10 µg AD0180 LANCE Eu-W1024 labeled phosphoserine/threonine-proline antibody (Upstate) 10 ug AD0182 LANCE Eu-W1024 labeled phospho-(thr) PKA substrate antibody (CST) 10 µg AD0184 LANCE Eu-W1024 labeled phospho-(thr) AKT substrate antibody (CST) 10 µg AD0186 LANCE Eu-W1024 labeled phosphoserine antibody (Zymed) 10 µg AD0188 LANCE Eu-W1024 labeled phospho (Ser) PKC substrate antibody (CST) 10 µg AD0190 LANCE Eu-W1024 labeled phospho (Ser) binding motif antibody (CST) 10 µg AD0192 LANCE Eu-W1024 labeled phospho-(ser) binding motif 4E2 antibody (CST) 10 µg AD0201 SureLight Allophycocyanin-streptavidin (APC-SA), protein binding assay 1 mg AD0202 SureLight Allophycocyanin-streptavidin (APC-SA), protein binding assay 30 mg AD0203 LANCE Eu-W1024 labeled anti-phosphotyrosine antibody (P-Tyr-100) 10 µg AD0205 LANCE Eu-W1024 labeled anti-his antibody 10 µg AD0206 LANCE Eu-W1024 labeled anti-c-myc antibody 10 µg AD0211 LANCE Eu-W1024 labeled Protein G 10 µg AD0212 LANCE Eu-W1024 labeled anti-human IgG 10 µg AD0245 TruPoint Caspase-3 Assay Kit 96 3x96-well plts AD0246 TruPoint Caspase-3 Assay Kit 96 w/o plates 288 assay points AD0252 LANCE Eu-W1024 labeled anti-gst antibody 10 µg AD0253 LANCE Eu-W1024 labeled anti-gst antibody 50 µg AD0254 LANCE Eu-W1024 labeled anti-gst antibody 1 mg CR Stabilizer (7.5%, DTPA-purified BSA) 50 ml CR LANCE Eu-W1024 biotin 10 nmol CR TruPoint Caspase-3 substrate 5x90 nmol CR TruPoint Caspase-3 reaction buffer 1 L CR TruPoint Caspase-6 substrate 5x90 nmol CR TruPoint Caspase-8 substrate 5x90 nmol CR LANCE detection buffer, 10x 250 ml CR SureLight Allophycocyanin-streptavidin (APC-SA) 1 mg CR SureLight Allophycocyanin-streptavidin (APC-SA) 50 mg PerkinElmer Life and Analytical Sciences 710 Bridgeport Avenue Shelton, CT USA Phone: (800) or (+1) For a complete list of global offices visit PerkinElmer, Inc. PerkinElmer is a registered trademark of PerkinElmer, Inc. FLAG is a registered trademark of Sigma Aldrich Co. SureLight is a registered trademark of Martek Biosciences Corporation. All other trademarks and registered trademarks are the property of PerkinElmer, Inc. PerkinElmer reserves the right to change this document at any time and disclaims liability for editorial, pictorial or typographical errors _01 ELEC0703XX