1.1.Primary objective is to determine the binding specificity of Glycan Binding Proteins (GBPs) and organisms submitted by investigators, using the printed glycan microarray platform.
3.1.Glycan printed slides, printed on the side of the slide with the white etched bar code and black marks- DO NOT TOUCH THIS AREA
3.2.Cover slips (Fisher scientific, 12-545F)
3.3.Humidified Slide processing chambers (Fisher scientific, NC9091416), or homemade system using Petri Dish, with wet paper towels in the bottom of the chamber
3.4.100 ml Coplin jars (3) for washing slides
3.5.Tris-HCl (Fisher scientific, BP152-1)
3.6.NaCl (Fisher scientific, S271-3)
3.7.CaCl2 (Fisher scientific, C79-500)
3.8.MgCl2 (Fisher scientific, BP214-500)
3.10.Bovine Serum Albumin (BSA), Protease Free, LY-0081 (Boval Company)
3.11.Tween-20 (EMD Biosciences, 655205)
3.12.GenePix 4300A scanner (Molecular Devices)
4.1 TSM = 20mM Tris-HCl, pH 7.4 150mM NaCl, 2mM CaCl2, 2mM MgCl2
4.2 TSM Wash Buffer (TSMW) = TSM Buffer + 0.05% Tween-20
4.3 TSM Binding Buffer (TSMBB) = TSM buffer + 0.05% Tween 20 + 1% BSA
1L 10X TSM Washing Buffer Stock Solution
0.20M Tris- HCl
1.5M Sodium Chloride (NaCl)
0.02M Calcium Chloride (CaCl2)
0.02M Magnesium Chloride (MgCl2)
Weigh out required amount of Tris-HCl and NaCl
Dissolve in dH2O and bring volume up to 800ml
pH solution and add HCL or NaOH to adjust pH to 7.4
Add appropriate concentrations of CaCl2 and MgCl2
Monitor pH while bringing up volume to 1000ml (1L)
Adjust if necessary
Filter to increase shelf lifespan
Store at Room Temperature (RT)
Add 10 ml of 10X TSM buffer to 100ml of dH2O
Add 20 g of Tween 20 to 100ml of dH2O
TSM Wash Buffer
Add 10 ml 10X TSM to 100ml of dH2O
Add 250 µl of 20% Tween-20 for final concentration of 0.05%
TSM Binding Buffer
For 100 ml:
1X TSM buffer
250 µl of 20% Tween-20
1 g BSA
5.1.Take out Reagents and bring to room temperature
5.1.1.Buffer (A) TSM
5.1.2.Buffer (B) TSMW
5.1.3.Buffer (C) TSMBB
5.2.Sample Preparation: Prepare 100 µl of sample by diluting the fluorescently labeled Glycan Binding Protein or Organism in TSMBB or appropriate Binding Buffer based on properties of GBP or Organism to an appropriate final concentration required for the analysis.
5.3.Hydrate slides in 100 ml of TSMW in a Coplin Jar for 5 min and drain excess buffer from slide by setting the slide upright on a paper towel and wipe the back of the slide with a Kim wipe.
5.4.Carefully apply 70 µl of sample to slide, in between the black marks.
5.5.Slowly lower cover slip onto slide, trying to avoid the formation of bubbles in the sample under the cover slip; carefully remove any bubbles by gently tapping the cover slip with a pipette tip, making sure the cover slip is between the black marks.
5.6.Incubate slide in a humidified slide chamber in the dark for 1 hr at RT.
5.7.After 1 hr incubation, remove cover slip by gently allowing it to slip off into the glass trash/biohazard trash and pipette a small amount of TSMW over the slide surface to remove the sample.
5.8.Wash the slide by gently dipping 4 times into 100 ml of each of the following buffers in Coplin Jars:
5.9.Spin slide in slide centrifuge for ~ 15 seconds or remove water under a gentle stream of nitrogen, and wipe the bottom of the slide to remove any droplets of water.
5.10.Scan in a fluorescent Scanner at the appropriate wavelength (See Scanning Protocol).
NOTE: Record any changes to procedure on Glycan Array Processing Form (GAPF).
Record Slide number on GAPF and record in personal slide worksheet on the server
***Some samples may require different buffers. ***