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Amount of GBP required for screening protocol on the printed microarray |
Investigator supplies Protein-Glycan Interaction Core 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, the Core 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.
Protein-Glycan Interaction Core 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.).
Protein-Glycan Interaction Core 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).
Protein-Glycan Interaction Core 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.).
Protein-Glycan Interaction Core 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 , prior
to sending lectins for analysis (special considerations on a case by case basis).
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