MATF Antigen Submission Details and Standard Project Deliverables

Transcription

1 MATF Antigen Submission Details and Standard Project Deliverables What we require when you submit your antigen: Proteins For a recombinant protein target, we require a minimum of 400µg soluble recombinant protein at a minimum of 250µg/ml. Nearly all tags are permissible, but some are very immunogenic and should be avoided (e.g. NusA), contact us to discuss if you are not sure. Common and acceptable tags are: GST, His-6 and MBP We will screen your custom monoclonal antibodies against the protein(s) you supply and also against an irrelevant protein with the same tag to ensure that the antibodies are recognising exclusively the target protein. Peptides Using a standard of suite of software, we can screen peptide sequences for you in order to select suitable candidate peptide antigens. We do not synthesise peptides ourselves but would be happy to use our preferred supplier and provide a quotation for the peptide synthesis, purification and coupling to a carrier protein. Purity Major contaminants must be removed from the protein samples used for immunisation. The protein should be as pure as possible. If necessary, you may wish to perform a double purification by running the protein over an affinity column, followed by gel filtration/size-exclusion chromatography. If the recombinant protein is insoluble, we will require 200µg of protein in a gel slice excised from an acrylamide gel (stained with Coomassie blue), and an additional small amount (at least 50µg) of the insoluble protein as an inclusion body pellet, including instructions for solubilising it in an appropriate buffer (e.g. 8M urea). Buffers Buffers for immunogens (to be injected) should be physiologically compatible (i.e. PBS) where possible. Buffers should be non-toxic and non-irritant (i.e. no azide and urea concentration must be less than 1M). What we deliver for a standard custom monoclonal antibody project: Unless otherwise specified, we will only deliver IgG-secreting clones. The standard service agreement includes up to 24 clones that produce antibodies that specifically recognise the target antigen used for immunisation. One of these cell-lines is subcloned by serial dilution (2 rounds) to ensure monoclonality. In addition to hybridoma cell lines, we will deliver up to ~5ml of culture supernatant for each of the isolated clones for testing and characterisation. Additional clones can be cultured upon request, but will incur an additional cost per clone. We bank a duplicate vial of each positive cell line isolated for a maximum period of 6 months. Project Timelines: MATF can usually complete a project to the 24 clones stage within weeks of the first immunisation. Subcloning usually takes an additional 4 weeks. Some projects may take longer if there is a problem eliciting an immune response. 1

2 MATF Project Submission Form Please enclose a signed HARD COPY of the completed form with your samples. Please ensure that you complete the form below in detail. The asterisks (*) indicate areas that are required to be filled. This will help us with the design of your project and ensure that we meet all your project requirements and achieve the best possible outcome. If you are having any difficulties populating this form, please notify us and we can arrange an appointment to complete the form. Alternatively, contact us at info.matf@monash.edu if you have any questions or queries. Confidentiality This project is classified as fee-for-service project and all information provided in this form below will remain strictly in confidence. The intellectual property rights attaching to all the material created and prepared by MATF in connection with the performance of their services shall vest in you (please refer to Terms and Conditions on on page 6 for more information). Contact Information *First Name: * Address: *Telephone: *Mobile: On-line quote reference: *Shipping Address: *Surname: *Group/Company/Institution: Fund Number: Cost Centre: Promotional Discount Code: Antigen Information (Immunisation Sample) *Antigen name: Short name (optional): *Type of antigen: Native Protein Recombinant Protein Peptide Carbohydrate Lipid Virus Bacterium Whole Cell Cell Lysate Other: *Concentration: mg/ml *Total amount provided: mg *Is your antigen toxic, or harmful to humans/animals: Yes No *Format: Liquid Lyophilized Gel slice Other: How to reconstitute if lyophilized? *Antigen species of origin: Human Mouse Rat Yeast E.coli Other: 2

3 *Does your antigen have a tag or conjugate? Yes No (Nearly all tags are permissible, but some are very immunogenic and should be avoided (such as NusA). If YES, please specify: KLH BSA OVA Other: *Is the sequence of the antigen available? Yes No If YES, please specify sequence: *Does your antigen have any known homologs in other species? Yes No If YES, please specify: Mouse Rat Other: Percentage homology (if known): % *Size of antigen (molecular weight, number of amino acids): *Size of antigen (separated by SDS-PAGE): Reduced gel: kda Non-reduced gel: kda *Please attach a picture of the acrylamide gel showing your antigen stained with Coomassie brilliant blue, (separated by reducing or non-reducing SDS-PAGE). 3

4 *Do you have any previous experience raising antibodies against this antigen? Yes No If YES, please include any relevant data and/or references about previous attempts. Project Requirements *Do you require additional subcloning?: Yes No To ensure monoclonality, subcloning of one clone is included with your standard project. For additional subcloning, we charge $880 per clone. *Do you require purified antibody?: Yes No MATF can grow and purify 1-100mgs of antibody. Additional costs apply. *Do you require IgM isotypes?: Yes No If YES, how many additional clones?: If yes, what amount is required (mg)?: MATF will typically only keep IgG isotypes unless specified. *Do you require isotyping? Yes No Note: There is a charge of $110 per clone. Antigen Storage/Shipping Conditions *What is the storage/shipping temperature? Room temperature 4 C -20 C -80 C If shipped in solution, what buffer is used? *Number of vials shipped: *Additional Screening Sample(s) Information MATF can screen against multiple peptide/protein samples. Please specify any additional proteins that will be included for screening, and ensure you state if the antibodies should bind these samples or not. 4

5 Please note: MATF will endeavour to provide clones that meet all of your requirements, however we can only ensure clones generated produce antibodies that bind the antigen. Sample name Concentration (mg/ml) Total amount (mg) Buffer Abs should bind/not bind to this sample? Storage conditions for this sample Key: Abs: Antibodies, Storage conditions: i.e. temperature, exact compositions of buffers, concentration of salt, ph etc. Fill in any specific requirements that should be noted. Project Design If you have any additional comments, requirements or other information you wish to provide, please write in the space below: Project Design Notes 5