Custom Polyclonal Anti-Peptide Antibody, Brochure

Transcription

1 Custom Polyclonal Anti-Peptide Antibody, Brochure Interest in any of the products, request or order them at Bio-Connect. Bio-Connect B.V. T NL +31 (0) T BE +32 (0) Begonialaan 3a F NL +31 (0) F BE +32 (0) TE Huissen E info@bio-connect.nl The Netherlands W

2 CUSTOM ANTIBODY PRODUCTION CUSTOM POLYCLONAL ANTI-PEPTIDE ANTIBODY Services : From single immunization to PACKAGE Service Personalised projects : Adapted protocols to your needs Technical support : Advice from experts in the field of immunology IMMUNOGEN Conjugation to carrier protein IMMUNIZATIONS Guinea Pig 81D 53, 67& 90D Example of immunizations using STANDARD PACKAGE (<16AA, >70% purity, 5-10mg) Conjugation to carrier protein (KLH, OVA, BSA) Immunization of 2 animals according to the protocol of your choice (<16AA, >70% purity, 20-25mg) Conjugation to carrier protein (KLH, OVA, BSA) Immunization of 2 animals according to the protocol of your choice ELISA test for each bleed Immunopurification of 5 ml of serum Shipping of the immunopurification s column

3 Immunogen To undertake the design of a peptide, the following information will be required: Protein name Sequence (FASTA format) by or computer compatible support (CD-ROM) if the project is confidential Domains of interest : N-ter or C-ter, cytoplasmic or extracellular Specificity required by comparison to other proteins List of homologous proteins Application : WB, IHC, IP... By , all peptides that we have selected, from which you can choose the peptides to be injected. Peptide synthesis You can use 1, 2 or 3 peptide(s) for your antibody development. However this possibility depends on the results of the peptide design. All peptides are produced with Fmoc strategy on solid phase and analysed by HPLC to ensure purity and by mass spectrometry to determine the molecular mass. The quantity to be synthesized depends on the service chosen: Standard Service : peptide <16 AA, 5-10 mg, >70% of purity, no modification Package Service : peptide <16 AA, mg, >70% of purity, no modification Nevertheless, you can choose to synthesize the peptides with other quantities, purities, sequence length We advise you. To co-inject 2 peptides during your project for the following reasons: More of the entire protein is used The immune system is even more stimulated You increase the sensitivity of detection You should know that we never inject more than 3 peptides as immuno-domination is frequently observed. For each peptide: All peptide remaining after injection A technical data sheet including the characteristics of your product Mass spectrometer and HPLC profiles Conjugation Usually we conjugate the peptide to a carrier protein via a cysteine residue. If the amino acid sequence of the peptide does not contain a natural cysteine, we add a cysteine to N-ter or C-ter sequence of the peptide. Carrier proteins available: Keyhole Limpet Hemocyanin (KLH), Bovine serum Albumin (BSA) or Ovalbumin (OVA) Quantity of peptide conjugated : 5 mg (Exception for goat projects : 10 mg will be conjugated).

4 Immunizations Animals Covalab can immunize the animal of your choice: rabbit, chicken, guinea pig, rat, mouse or goat. Animal Guinea Pig Origin New-Zealand Isa-Brown Dunkin Hartley Sprague Dawley Alpine Chamoisée BALB/C We advise you To develop your antibodies in rabbits as this animal provides the best ratio in terms of immune response and quantity of serum. To inject at least 2 animals in order to increase the chance to have a good immunoreactivity Our Protocols We provide a large range of protocols that can be adapted to your needs. At the end of the standard protocol, you can request: - The final bleed of your animals - One or several additional bleeds to obtain greater volume of serum - To continue immunization in order to improve the serum s immunoreactivity Protocols Steps 53D 81D Guinea pig 90D Animal transfers to the experimental zone to -3-6 Pre-immune bleed & First injection Second injection (1 st boost) Third injection (2 nd boost) Bleed Fourth injection (3 rd boost) Fifth injection (4 th boost) Bleed End of the standard immunization protocol Volumes of serum (per animal) 53 D 67 D 88 D 90 D Guinea Pig 88 D 81 D 90 D Volumes 1 st Bleed 4-5 ml 4-5 ml 4-5 ml 1-2 ml 0,5-1 ml 0,5-1 ml 5 ml 2 nd Bleed ml 4-5 ml 4-5 ml 1-2 ml 0,5-1 ml 0,5-1 ml 20 ml 3 rd Bleed ml ml 1-2 ml 0,5-1 ml 0,5-1 ml 200 ml Final Bleed ml ml ml ml 4-8 ml 4-8 ml 1000 ml 15-30µl 15-30µl µl A detailed protocol of your study describing the different injections, bleeds and shipments

5 Related Services Animals selection If you wish to select the best rabbits for your project, we can send you pre-immune bleeds for testing. ELISA Anti-peptide Each sample will be tested against each peptide(s) by ELISA. These ELISA tests will allow you to follow the evolution of the immunoreactivity of the rabbits during the immunization. For each test, a data sheet showing the serum s titers and the evolution of each animal s immunoreactivity. These elements and the results obtained in your lab will allow you to take the best decision concerning the future of your project. D O à 450 nm 3 2,5 J0 J53 2 J75 1,5 1 0, Figure: Example of curves provided to our customers, showing the immunoreactivity of each bleed 1/Dilution Purifications Immunopurification of Anti-peptide antibodies This type of purification will allow to obtain the antibodies specific to the peptides. This will decrease the background during your experiments. This service consists of : Conjugation of the peptide(s) on sepharose Passage of the serum on the column You will be able to choose the following volume: 5, 10, 25, 50 or 100 ml of sera Elution of immuno-adsorbed antibodies Control of the immunoreactivity of the antibodies by ELISA Note : The immunopurification of 5ml of serum is included in our PACKAGE service Please note The purified antibodies are provided in Tris 1M ph 8, Azide 0.02% Buffer. (If you do not wish azide to be added, please notice us on your order) The average concentration for serum immunopurification is from 50 to 100 µg of Antibody/ml (This is just an indication; and can not be guaranteed) Your purified antibodies and an information sheet showing a report of your purification and the antibody concentration Purification of IgY from egg yolk This service includes a purification of IgY from egg yolks by selective precipitation, an ion-exchange chromatography. (Carroll SB and Stollar BD. Antibodies to Calf thymus RNA polymerase II from egg yolks of immunized hens.(1983) J Biol Chem Jan 10;258(1):24-6). CovalAb Tel : +33 (0) Fax : +33 (0) enquiries@covalab.com - web : CovalAb UK Tel : +44 (0) Fax : +44 (0) enquiries@covalab.co.uk - web :