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GLYCAN ARRAY SCREENING

GBP Preparation for Analysis

 
Amount of GBP required for screening protocol on the printed microarray (v2.1) Investigator supplies Core H with 20 to 40 micrograms of purified GBP.
Number of glycan targets: 285
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
 
Amount of GBP required for screening protocol on the 384-well microplate-based glycan array (v3.8) Available at discretion of Core H based on resources available.
Investigator supplies Core H with 1 to 2 milligrams purified GBP and sufficient amounts of appropriate detection reagents.
Number of unique ligands: 230
Replicates: 3
Positive Controls: 3
Negative Controls: 3
Volume per well: 50 microliters
Total volume GBP required: 35.4 milliliters
GBP concentration: 30 micrograms/ml
Total FITC-labeled GBP or Ig chimera required per primary screening: More than 1 milligram
 
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.
 
Ig-chimera analysis using FITC-labeled secondary antibodies
Investigator specifies primary host or Ig used.
Core H will provide Alexa-labeled secondary antibody.
 
Recombinant tagged or fusion GBPs
Investigator specifies construct of fusion protein or tag (GST, His, S-Tag peptide).
Core H will provide appropriate labeled, commercially available secondary antibody.
 
Anti-glycan antibodies
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.
 
Special conditions for the following:
GBPs detected by secondary antibody and labeled anti-immunoglobulin, GBPs precomplexed with antibodies, cells, virus, or bacteriophage
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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Last Updated Wednesday, 18-Oct-2006 13:38:00 EDT. Please contact us with comments/questions.