Protocol ID: Printed Array Screening
Title
:
Printed Array Screening
Category
:
Lectin-Glycan Binding
Overview
:
Protocol for printed array from Core H.
Procedure
:
Glycan array Screening Protocol for the Consortium Printed Array
Slides: Schott Nexterion H slides (Schott Cat. No. 1070936B)
Glycan spotting concentration: 100uM
Glycan Replicates: n=6
Slides are printed at the Consortium Carbohydrate Synthesis/Protein Expression Core D located at the Scripps Research Institute.
For more detailed information about the printed array, please contact Core D Director:
Ola Blixt, Ph.D.
olablixt@scripps.edu
Or refer to:
Blixt et al, Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. PNAS 2004 Vol. 101, no. 49, pp17033-17038.
Slide Conditioning
1. Preprinted slides are soaked in deionized water for 5 minutes at room temperature and dried under a stream of nitrogen.
Assay Buffers
TSM Buffer: 20 mM Tris-Hcl, pH 7.4 (or MOPS 20mM)
150 mM NaCl
2 mM CaCl2
2 mM MgCl2
TSM Wash Buffer: TSM buffer + 0.05% Tween 20
TSM Binding Buffer: TSM buffer + 0.05% Tween 20 + 1% BSA
Direct Binding Assay ýV Lectin labeled with FITC (Alexa 488)
1. Lectin is diluted to assay concentration in binding buffer.
2. 50 microliters of labeled lectin is applied to the printed surface and coverslipped.
3. Slide is incubated protected from light in a humidified chamber for 1 hour at RT
4. Slide is removed, coverslip removed and washed for 10 seconds each in wash buffer, wash buffer lacking Tween 20, and deionized water. The slide is dried under a stream of nitrogen.
5. The binding image is read in a Perkin Elmer Microscanarray XL4000 scanner and tiff file of image stored.
6. Image analysis is performed using Imagene (V.6) image analysis software.
7. Raw data results files are generated in Excel format and uploaded to the Consortium database as .dat files.
8. Imagene data is uploaded to the database as .txt files and
Assays detected with FITC labeled 2o Ab
Requires an additional incubation step and wash
1. After step 3 above, the slide is washed 1x in wash buffer, 50ul of the appropriate 2o Ab applied and coverslipped and incubate 1 hr at RT
2. Proceed to step 4 above.