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2 9 Affinity Purification Custom Peptide Synthesis Polycolonal Antibody Production Monoclonal Antibody Production 1-D & 2-D Gel Analysis Recombinant Technologies (visit for more custom lab services) > accelerate: lifescienceresearch

3 Affinity Purification Antibody Purification with Protein A bioworld can purify your monoclonal and polyclonal IgG from ascites and serum. Ideal for subclasses of IgG from human, mouse, rabbit and sheep. bioworld can purify your monoclonal and polyclonal # service inquire Antibody Purification with Protein G IgG from ascites, serum and cell culture supernatants. Ideal for IgG subclasses which bind poorly to protein-a such as bovine, goat and rat. Antigen Affinity Purification bioworld can perform custom coupling of an antigen or # service inquire peptide of your choice to an affinity matrix and use it to purify your antibodies. Standard coupling chemistry Removal of. coli- Contaminant Antibodies To remove contaminant antibodies from a protein Affinity Purification of Ab bioworld can perform affinity purification to make your # service inquire expressed and produced in. coli and used as an antigen for antibody production. Standard coupling chemistry sample free of other contaminating antibodies deliver peptide-specific antibodies. # service inquire # service inquire FlexArm Linked Affinity Matrix bioworld can link your peptide # service inquire (may be submitted or produced by bioworld) to a bioaffinity matrix using flexarm linker to provide hydrophilic spacer and acheive highly efficient matrix binding. See FlexArm peptide synthesis for more information, page 134. Ubiquitin-Binding Protein Pufification We can purify ubiquitinylated # service inquire protein/enzymes from your sample using ubiquitin affinity chromatography. Useful for ubiquitin activation enzyme (1), ubiquitin carrier enzymes (2s), ubiquitin ligases (3 s) and ubiquitin terminal hydrolyases (ucms). Calmodulin-Binding Protein Purification bioworld purify calmodulin # service inquire can binding proteins using calmodulin affinity chromotography. These proteins can be further analysed and identified using MASS spec. Cleaning of Specific Antibodies bioworld labs provide affinity purification services by using a unique peptide-agarose coupling using a flexible arm PG flexarm linker to allow better binding of antigen to yield highly specific polycolonal antibodies. Per 1ml serum # KLH $ To deplete KLH Abs Per 1ml serum # Coli $ To deplete. Coli Abs depletion Affinity Purification Matrix Ligand bioaffinity Matrix Protein Impurities lution Buffer 131 Pure Protein

4 Custom Peptide Synthesis Custom Peptide Synthesis bioworld s Custom Peptide Synthesis service is perfect for any research facility looking for the highest quality, low cost service available. For unique & larger peptides, as well as specific peptide modifications, please contact us. You can use the Peptide Synthesis and Antibody Production Quote forms. or or call bio.PLUS. Antigenic pitope Analysis (*free) Antigenic analysis of a protein sequence to determine the most antigenically unique regions of the protein. # service $ Results can be used for further peptide synthesis & antibody production. * If peptide synthesis or antibody production is contracted through bioworld or Peptidexpress, the analysis fee is waived. Computational Analysis The latest software is used to analyze your DNA or protein sequence to project the physical characteristics of the expressed protein including extinction coefficient, isoelectric point, charge, hydrophobicity, & secondary structure. BLAST is performed to compare your sequence to published sequences to determine common & species-specific regions to aid with the design of peptides. Identify various protein motifs to aid in the identification of your protein function. A complete restriction site analysis is performed to aid in vector construction. # service $ A search for silent mutations that result in the appearance or disappearance of restriction sites without changing the protein sequence. Oligonucleotides for PCR amplification, sequencing primers, & hybridization probes are designed & analyzed for melting temperatures. Peptide Synthesis and Purification The peptides are produced using solid phase peptide synthesis. Fmoc is utilized as the amino protecting group & TFA is employed to cleave the peptide. The peptide is delivered unpurified ( 65% purity) and desalted but can be furthur purified to suit your research applications. Purity of all bioworld s peptides are tested using HPLC & MASS Spectrometry. The price per residue is for peptide sequences 8-20 amino acids in length. Peptides having less than 8 or more than 20 residues will have a slightly higher cost. Discrete PG (polyethylene Glycol) residues can be incorporated in a peptide to increase its solubility both to increase its bioactivity and further linkage with any carrier protein to enhance antigenicity. (see page 134) Synthesis of 8-30 residues # per residue $ (desalted) 70% Purity # process $ mg peptide yield. 80% Purity # process $ mg peptide yield. 95% Purity # process $ mg peptide yield. For quotes & order forms, please visit: # 132 > accelerate: molecularlifescienceresearch

5 Custom Peptide Synthesis (contd.) Peptide Modifications Acetylation Amidation (C-termiinal) Anilide (C-terminal) Benzyloxcarbonylation Biotin Dansylation # $ # $ # $ # $ # $ # $ MAP (multiple antigenic peptide) System A revolutionary antigen system for high titer specific antibody production without conjugation to carrier proteins. Allows for production of highly efficient immunogens. Peptides can be synthesized on a symmetric or asymmetric lysine core with amino terminals (branches) available for peptide attachment. Responses to MAP antigens are usually quick & potent, yielding higher titer than those produced by hapten-carrier conjugates. Peptides >15 amino acids yield better antibody production. MAP system set up fee is required for all MAP peptides. Dinitrobenzolylation (DNP) Fatty Acid Formylation Nitronation Phosphorylation - Serine Phosphorylation - Threonine Phosphorylation - Tyrosine Succinylation Sulfonation Fluorescene/Dye Labeling # $ # $ # $ # $ # $ # $ # $ # $ # $ Set Up Fee # fee $ MAP Synthesis # per residue $ Asymmetric # branches $ # branches $ Symmetric # branches $ # branches $ For hydrophylic MAPs see page 134 Custom Peptide Synthesis Aminocoumarin # $ Fluorescein # $ Lissamine Rhodamine # $ NBD # $ Tetramethylrhoddamine # $ Custom modifications & conjugation services available. Contact: tech@bio-world.com (Note: MAPS can create artifical antigenic epitopes) see fleximap, page

6 Custom Peptide Synthesis FlexiPep Service Peptides are synthesized on the ends of flexible dpg spacer # service inquire arms. The spacer arms remain attached to your peptide after synnthesis, increasing the solubility of the peptide. This also provides a point of attachment for conjugation to other ligands. FlexiPep peptides are more antigenic. FlexiMAP Service NH-Fmoc - Peptide Peptides are synthesized on the # service inquire ends of flexible dpg spacer arms. When these complexes are cleaved from their stationary phase, they produce a highly soluble multi-antigenic peptide. FlexiMap offers enhanced antigenic displays without the need for conjugation to a carrier protein. This eliminates unwanted epitopes, creating a more precise immune response and produce specific antibodies. HO d(pg) 4 Peptide Synthesis and Antibody Production The project includes: Peptide # rabbit $ Synthesis from 8-20 amino acids, KLH conjugation of the # rabbits $ peptide, Ab Production. Protocol is approximately 90 days-including Peptide Synthesis & Ab Production, a positive control bleed, three test bleeds & boosts, a production bleed & a final exsanguination bleed. The project is delivered in 2-3 shipments, (shipped on dry ice, costs are extra) ach Peptide Synthesis & Ab Project is held in strict confidence & complete records provided upon completion. The antisera can be further purified and or lypholized for overseas shipments. Peptide dpg Conjugation: KLH / BSA bioworld offers a unique peptide KLH/BSA conjugation service using flexible arm linker of discrete size polyethylene glycol with NHS. Such conjugations are often more immunogenic and specific because of the high solubility of the peptide and flexibility of the antigenic epitope with a hydrophylic spacer arm. KLH Conjugation # NHS dpg $ NH-Fmoc - Peptide BSA Conjugation # NHS dpg $ enhance antigenicity of your peptide epitope Protein lectroelution for Antibody Production Send your sample and we can # elution $ run a SDS gel and electroelute your protein band for antibody # D gel/elution $ production. > 134 accelerate: proteinresearch

7 Polyclonal Antibody Production Service bioworld produces high quality antibodies at costs substantially lower than most laboratory facilities. For a Custom Lab Service Quote, access and fill out the Antibody Quote Form at or call bio.PLUS. The quotation will include the following: all costs, schedule and delivery of this final product. Primary antibody Substrate Secondary antibody Primary antibody Substrate Biotinylated primary antibody Substrate Substrate Substrate Substrate Ag Ag Ag Direc Assay Indirect Assay Srtaptavidin-Biotin Complex bioworld offers many hosts suitable for polyclonal antibody production including rabbits/ guinea pigs/ mice/ chickens/ goats/ sheep,/and horse. Typically, 1mg of antigen is required for 1 rabbit. Smaller can be administered but the immune response of the host may be less potent. Antigens can be provided to bioworld in a detergent-free buffer solution, as a lyophilizate, or a crushed polyacrylamide gel. ach Polycolonal Antibody project is held in strict confidence. bioworld will not accept antigens that are pathogenic, toxic and/or carcinogenic. Rabbit # process $ Host Procurement & conditioning # process $ Pre-inoculation Bleed # process $ Antibody Schedule Day 0 Day 21 Day 35 Day 49 Day Day >90 Pre-inoculation and initial Inoculation First test bleed and boost (using complete Freund s Adjavent) Second test bleed and boost Third test bleed and boost Production Bleed (Included in porject cost) and Final xsanguination Bleed (available at additional cost) xtended protocols (xtended protocols will have additonal fees) Antibody Production Service Host Disposal # process $ Shipping (dry ice) $ / shipment bioworld offers many additional services to enhance your Polycolonal Antibody Project including: LISA analysis of the test bleeds, antibody purification using Protein A or ammonium sulfate fractionation lyophilization of antiserum and prolonged host maintanence. 135

8 Antibody Production Service Monoclonal Antibody Production Service bioworld s custom monocolonal antibodies production service against your antigen, includes development of primary antibodies, fusion and ascites production. Other services required for screening of hybrids and selection of clones can be requested by the researcher as needed. ach Monocolonal Antibody project is held in strict confidence and complete record is provided at the conclusion of the project. Development of Primary Antibodies # $85.00/mouse LISA screening to select host for Hybridoma # $150.00/mouse Fusion and development of hybridoma # $ 2, LISA screening for selection of clones Cloning of selected hybrids # $ LISA screening of clones xpansion of selected clones # $ # $ # $ Monoclonal Antibody Production Project Phase 1 # service $ 1, Step 1: Immunization Immunization of 5 Balb/c mice. In general we begin with a priming dose of 50 to 200 µg followed by several boosting doses each containing 50 to 200 µg. This regime will continue until a sufficient antiserum titer is achieved. Step 2: Screening Test bleeds are evaluated by using an indirect LISA. Optimal LISA conditions are developed to ensure the proper evaluation in other assays. Step 3: valuation Data is evaluated to determine the performance of antiserum. Antiserum titers and specific assay performance must be acceptable to continue project. Phase 2 # service $ 3, Step 4: Hybridoma Fushions A splenocyte fusion is performed on host animals with the best response as determined by LISA or other assay. The lymphocytes are then fused to an Sp2/0-Ag14 cell line at a 5:1 ratio using the PG method. Step 5: xpansion & Screening The fusion is plated into 24 or 96 well plates. Plates are monitored for growth and fed weekly. Wells with cell growth are screened by LISA and/or other methods at days after fusion. Ascites production <100ml # $ 9.50/ml Phase 3 # service $ 2, Ascites production >100ml # $ 7.60/ml > accelerate: monoclonalantibodyproduction Step 6: Subcloning Cells from up to 5 strongly positive parental clones from fusion wells are expanded, isotyped and subcloned by limiting dilution. More subclones can be processed at your request for additional charge. Step 7: xpansion & Screening Subclones are screened by LISA, positives expanded, and screened again. Up to (2) positive subclones from each parental subclone will be expanded, frozen, and stored for up to six (6) months.

9 1-D & 2-D Gel Protein Analysis 1-D Gel Service lectrophoresis 1-D SDS gel running service. Acrylamide gel % indicated at the customer s request Option of 10, 12, wells per comb Large size: 16 x 13cm, small 8 x 8cm Silver Stained We provide fully documented results with a publication quality print & stained gel (wet or dry). Special handling of the gel is required to prevent degradation of proteins in order to get reproducible results (call for details). Please call for instructions if shipment is radioactive (there is an ` additional disposal fee). Gel dimensions: 16 x 13cm A ph gradient blot is provided along with a description of method & gel loading information (custom ph gradients are available). Standard IF tube gel contains 2% (ph 4-8 BDH) ampholines. Molecular weight & internal standard marker are included in each gel. 10 times more sensitive than Coommassie Blue staining (depending on the protein). Gives qualitative rather than quantitative results. NOT: Silver staining is not compatible with mass spectrometry. A special silver stain can be substituted if mass spectrometry analysis will be performed. Please indicate when ordering. Find your spot # large $ # small $ # D gel $ D Gel Service Multi-step process used to resolve complex sets of proteins from total soluble or membrane protein fractions. Ideal for detecting enzyme isomers, protein phosphorylation, protein glycosylation, amino acid microsequencing, enzyme/protein induction studies, mutations & other protein modifications as well as a complete proteomics profile. bioworld provides fully documented results with a publication quality print & stained gel (wet or dry). A sample preparation kit (cat# ) is also available to ensure your protein samples are best suited for 2-D Gel lectrophoresis. Avoid degradation of proteins in order to get reproducible results. Gel dimensions: 16 x 13cm. A ph gradient blot is provided along with a description of method & gel loading information (custom ph gradients are available). Standard IF tube gel contains 2% (ph 4-8 BDH) ampholines. Molecular weights & internal standard marker are included in each gel. Coomassie Blue Staining PVDF Reveals the major proteins in # process $ the 2-D patterns & is necessary to visualize proteins for N-terminal sequencing. Useful when matching the blot to a duplicate gel & identifying proteins for Mass Spectroscopy analysis. The stain disappears during western blotting so a color electronic photo should be obtained before analysis. Coomassie Blue Stained # sample $ (See above description). # sample $ # sample $ Photography/Digital Image # process $ xcellent color images in.tif or.jpeg format. Price includes an image file on a CD & prints, or the file can be ed directly to you as requires. 1-D & 2-D Gel Analysis > accelerate: proteinresearch 137

10 Recombinant Technologies Sample Preparation Kit # Kit $ Provides necessary buffers (SDS, Urea, Lysis, Protease Inhibitors, & Nucleases) & components to adequately prepare samples for custom electrophoresis services. Adequate material to prepare 10 samples. Buffers must be shipped on dry ice. Sample Preparation Service # service $ Silver Stained 2D gel # sample $ (See Silver Stained under 1-D). # sample $ # sample $ Unique Spot Analysis # process $ bioworld provides detailed analysis for identification of unique spots in 2-D gels to identify a new gene product by comparing two samples. *The gels to be analyzed must be run by bioworld.* Custom IF or NPHG Isoelectrofocusing or # process $ non-equilibrium ph gradient electro-phoresis Used to resolve complex sets of proteins from total soluble or membrane protein fractions. bioworld provides fully documented results with publication quantity print and stained gel (wet or dry). Molecular weight and internal standard markers are included. bioworld can prepare the sample for you. for a fee of $ Transblotting onto Polyvinyl difluoride (PVDF) PVDF is directly compatible with # process $ protein gel sequencers & binds proteins more tightly than nitrocellulose giving greater recoveries. Carried out overnight according to standard procedures using a tris/ glycine/methanol buffer system without SDS. The dried unstained blot is returned to the client unless BP-2 or processed for Mass Spec analysis. Custom blotting services are available upon request, please inquire to change the procedure to your specifications. MALDI-TOF Mass Spectrometry Analysis Analysis of a peptide in solution # sample $ or eluted from gel (1-D or 2-D). Raw data is provided for # samples $ further analysis & interpretation. Data analysis available for an additional cost (please call for pricing) LC-MS/MS Protein Identification Service Protein identification service # sample $ includes sample preparation, digestion, peptide extraction # samples $ from 1D or 2D gel, nano- LC-MS/MS analysis and database interrogation. Duplicate Gel Service 1 duplicate of any gel Price per duplicate. # gel $ # gels $ # gels $

11 Recombinant Technologies Vector Construction Provide the gene insert either as purified DNA, within a purified plasmid or contained in a slab culture and bioworld will excise the insert and ligate it into an appropriate bacterial expression vector. The experimental design of the ligation may be facilitated using the latest software to design new recombinant DNA molecules or recreate gene segments using oligos with appropriate restriction sites and features that promote or enhance expression. Transformation of. coli using the subcloned plasmid will be performed. Colonies will be screened using conventional methodologies. Slab cultures require plasmid isolation prior to subcloning. Site Directed Mutagenesis Single mutation per plasmid Gene mutation, codon modification, or restriction site modification on any vector that is provided as plasmid DNA. # service $ # service $ Genomic DNA Library Construction A genomic library is constructed # service $ 3, in lambda phage or MBL vector. The DNA supplied is partially or fully digested with either co RI or Sau 3A-I, size fractionated and ligated into lambda phage vectors of choice, packaged and titrated. A library of 10 6 independent recombinants is provided. This library is amplified by infecting respective host strains of. coli. DNA Sequencing DNA sequencing is conducted # per reaction $ 6.50 by our highly trained and experienced staff with the Sanger s dideoxy sequencing protocol using ABI s automatic sequencer. bioworld will synthesize internal primers or subclone the fragment, if needed, or will purify the plasmid DNA for sequencing purposes. Both strands of DNA are sequenced depending upon the need of the investigator. Price includes one reaction for one sequence which equals,/a minimum of 500 base pairs. Minimum charge $100/projects cdna Library Construction Recombinant Technologies Baculovirus Cloning xpression A DNA insert from the vector # service $ provided is isolated and subcloned into a Baculovirus expression vector. The recombinant plasmid is transfected into SF9 or SF21 insect cells along with linearized wild type insect virus. The gene product of interest is identified by western blot analysis (if the antibody is provided) for the expression of the recombinant protein. A high titer virus stock in 50ml medium is provided to researcher for future studies. bioworld is able to create a # service inquire cdna Library of your choice, starting from 15µg of mrna. The library is produced in co RI or Xho I site of the vector. The cdna Library is made after 2 rounds of normalization (COT 100) thus enabling equal representation of low and high abundance cdna clones. The library provided contains 10 6 primary recombinants in the selected vector. bioworld requires at least 1mg total RNA or 10µg of poly (A+) RNA. The library will be ready for transfection purposes 139

12 mrna Isolation High quality mrna for # Sample <10g $ Northern blot analysis or cdna synthesis is isolated by oligo (dt) cellulose chromatography. To ensure the quality of the mrna, a photograph of the gel is provide for assurance. Northern Blot Analysis Northern blot analysis is performed with total RNA # analysis $ isolated from multiple samples with a single probe. The probe is isolated and hybridized to produce a publication quality blot. Southern Blot Analysis This service is performed by either single enzyme digestion # analysis $ of multiple samples or multiple enzyme digestion of the same sample. Our staff will be pleased to discuss this service in detail in order to determine the appropriate protocol suitable to your needs. Bio-World pitope Tagging Service pitope tagging eliminates the laborious and time-consuming task of producing a new antibody every time a different protein is studied. Bio-world provides custom pitope tagging with commonly used epitope sequences such as the influenza hemagglutinin protein (HA), human c-myc protein, six histidine residues (HIS6) and others, which can be easily inserted at the desired position within the protein coding sequence by recombinant DNA technologies. In particular, our Anti-HA Antibody High Affinity represents a powerful antibody for the detection and purification of HA tagged proteins due to its high affinity. Bio-World custom tagged proteins and anti-tag antibodies simplify the detection, characterization, and purification of proteins. S-Tag-pancreatic ribonuclease # service inquire (KTAAAKFRQHMDS) See Anti-S-Tag, pg 115 HA-Tag-influenza hemagglutinin # service inquire (YPYDVPDYA) See Anti-HA-Tag, pg 115 His 6 -Tag-Six histidine residues # service inquire See Anti-His 6 -Tag, pg 115 FLAG-Tag-Synthetic # service inquire (DYKDDDDKC) See Anti-FLAG-Tag, pg 115 C-myc-Tag # service inquire (QKLISDL) See Anti-C-myc-Tag, pg CustomFormulations Proteinxpression ProteinPurification for protocols visit