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Amount of GBP required for screening protocol on the printed microarray (v4.0) |
Investigator supplies Core H with 20
to 40 micrograms of purified GBP.
Number of glycan targets: 442
Replicates: 6
Positive Controls: 1 (Streptavidin)
Negative Controls: Determined by software
Volume per microarray: 70 microliters
Total volume GBP required: 200 microliters
GBP concentration: 200 micrograms/ml
Total FITC-labeled GBP or Ig chimera required per primary screening: Approximately 40 micrograms
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FITC-labeling of GBPs |
Investigators are expected to provide fluorescently labeled GBP. If the PI is unable to label the GBP, Core H at its discretion (based on resources available) can provide this service using the Alexa Fluor 488 Protein Labeling Kit (A-10235) from Molecular Probes.
Minimum requirement: 0.5 milligram protein either lyophilized or in 0.25 milliliters of an appropriate buffer. The GBP will be labeled and purified per kit manufacturer's protocol
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Ig-chimera analysis using FITC-labeled secondary antibodies |
Investigator specifies primary host or Ig used.
Core H will provide Alexa-labeled secondary antibody.
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Anti-glycan antibodies
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Investigator provides monoclonal antibody and specifies origin (species) and type (IgG, IgM, etc.).
Core H will provide appropriate labeled, commercially available secondary antibody.
Use of polyclonal serum is discouraged due to high backgrounds from endogenous anti-glycan antibodies present in most animal sera.
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Recombinant tagged or fusion GBPs
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Investigator specifies construct of fusion protein or tag (GST, His, S-Tag peptide).
Core H will provide appropriate labeled, commercially available secondary antibody.
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Anti-glycan antibodies
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Investigator provides monoclonal antibody and specifies origin (species) and type (IgG, IgM, etc.).
Core H will provide appropriate labeled, commercially available secondary antibody.
Use of polyclonal serum is discouraged due to high backgrounds from endogenous anti-glycan antibodies present in most animal sera.
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Special conditions for the following:
GBPs detected by secondary antibody and labeled anti-immunoglobulin, GBPs precomplexed with antibodies, cells, virus, or bacteriophage
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Investigators are to contact
David Smith, Director of Core H , prior
to sending lectins for analysis (special considerations on a case by case basis).
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