1. Introduction
1.1 Primary objective is to determine the binding specificity of directly labeled Glycan Binding Proteins (GBPs) submitted by investigators. The primary array platform is the CFG printed defined glycan microarray. Subsequently, remove binding using the MAWE method for future reuse of the slide.
2. References
2.1 www.functionalglycomics.org
2.2 Akul Y Mehta, Catherine A Tilton, Lukas Muerner, Stephan von Gunten, Jamie Heimburg-Molinaro, Richard D Cummings, Reusable glycan microarrays using a microwave assisted wet-erase (MAWE) process, Glycobiology, Volume 34, Issue 2, February 2024, cwad091, https://doi.org/10.1093/glycob/cwad091.
3. Materials Needed
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 SecureSeals or Proplate Chambers (Grace Biolabs, RD501388)
3.3 Aspirator
3.4 Orbital Shaker
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
3.9 dH2O
3.10 BSA (Fisher scientific, Bp1600-100) or Boval Bovine albumin, protease free (LY-008)
3.11 Tween-20 (EMD Biosciences, 655205)
3.12 Glycerol (ThermoFisher, G5516)
3.13 Sodium Azide (Fisher Scientific, S227-500)
3..15 Sodium Dodecyl Sulfate ( Sigma-Aldrich, L3771-100)
3.14 ImageXpress Pico (Molecular Devices, LLC) or similar scanner
3.16 Directly fluorescently labeled sample
4. Buffers
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
4.4 TSM Preservative Buffer (TSMPB)= TSM Buffer + 0.05% Tween-20 + 0.2% Sodium Azide + 5% Glycerol
4.5 10% Sodium Dodecyl Sulfate = 10% SDS
Buffer Preparation
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 4C
1X TSM- 50mL
Add 5ml of 10X TSM buffer to 45ml of dH2O
20% Tween-20
Add 20g of Tween 20 to 100ml of dH2O
TSM Wash Buffer-50mL
Add 5ml 10X TSM to 45ml of dH2O
Add 0.125ml of 20% Tween-20 for final concentration of 0.05%
TSM Binding Buffer- 50mL
1X TSM buffer
0.125ml of 20% Tween-20
0.5g BSA
100ml TSM Preservative Buffer
100 ml of 1X TSM buffer
0.25ml (250ul) of 20% Tween-20
5ml Glycerol (make sure to cut pipette tip when pipetting viscous liquids)
10ml 2% Sodium Azide
Store at room temp. in SL-409
5. Protocol
5.1 Take out Reagents and bring to RT- Aliquot into 50mL conical tubes
5.1.1 Buffer (A) TSM
5.1.2 Buffer (B) TSMW- Wash Buffer
5.1.3 Buffer (C) TSMBB- Binding Buffer
5.1.4 Buffer (D) TSM PB- Preservative Buffer
5.1.5 dH2O
5.2 Remove slide from fridge, rinse with MilliQ water, and spin dry in the slide centrifuge.
5.3 Attach SecureSeal to the slide and add 400 µL of TSMW buffer to the slide. Incubate for at least 5 minutes.
5.4 Scan the slide wet at the previous wavelength to ensure there is no residual binding
5.5 Sample Preparation
5.5.1 Prepare 400 µl of sample by diluting the fluorescent labeled Glycan Binding Protein or Organism in TSMBB or appropriate Binding Buffer based on properties of GB, or Organism to an appropriate final concentration required for the analysis. Refer to the Glycan Array Processing Form for specifics.
5.6 Aspirate TSMW from the slide. Add 400 µL of diluted sample to the slide.
5.7 Incubate away from light for 1 hour on the orbital shaker.
5.8 After 1 hr incubation, aspirate sample.
5.9 Wash the slide 4x with Buffer A (TSMW)
5.9.1 Repeat with 4x Buffer B (TSM), leaving the 4th wash on the slide.
5.10 Scan wet at the appropriate wavelength for the labeled sample (See Scanning Protocol)
6. MAWE Protocol
6.1 After scanning, wash the slide 2x with 10% SDS buffer
6.1.1 Keep the time between a completed assay and performing MAWE as short as possible. This ensures the MAWE protocol will effectively remove binding
6.2 Place in a glass container or petri dish and microwave on high for 3 seconds
6.3 Aspirate SDS, and add fresh buffer. Repeat 10x replacing buffer in between each microwave
6.4 Wash the slide 2 more times with 10% SDS buffer, then wash as in steps 5.8 and 5.8.1., and a final wash with water.
6.5 Place slide into a slide tube with TSM Preservative Buffer
6.5.1 Store at 4C until the next use
6.6 If using the slide immediately for another assay, scan the slide. If not, refer to step 5.4.
NOTE: Record specific details of the protocol on Glycan Array Processing Form (GAPF) that is specific to each sample set.
Record the Slide number on the GAPF
***Some samples may require different buffers or conditions. Always refer to the GAPF for investigator details.***