1. Introduction:
1.1. Primary objective is to determine the binding specificity of a Monoclonal Antibody submitted by investigators, using the printed glycan microarray, and removing with MAWE protocol for reusability.
2. Reference:
2.1. https://research.bidmc.org/ncfg
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. SecureSeal or Proplate Chambers (Grace Biolabs, 612202, 246868)
3.3. Orbital Shaker (Belco Biotechnology)
3.4. Tris-HCl (Fisher scientific, BP152-1)
3.5. NaCl (Fisher scientific, S271-3)
3.6. CaCl2 (Fisher scientific, C79-500)
3.7. MgCl2 (Fisher scientific, BP214-500)
3.8. dH20
3.9. Appropriate secondary antibody, fluorescently labeled
3.10. Bovine Serum Albumin (BSA), Protease Free, LY-0081 (Boval Company)
3.11. Tween-20 (EMD Biosciences, 655205)
3.12. Pico XPress (Molecular Devices)
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
Comment: For buffer preparation see Direct Glycan Binding Assay protocol: 4. Buffers
5. Protocol:
5.1. Make working stocks of washing buffers (TSM, TSM Wash Buffer, and H2O) or collect reagents and bring to room temperature if they have been in the refrigerator.
5.1.1. Buffer (A) TSM Wash Buffer (TSMW) - TSM Buffer + 0.05% Tween-20
5.1.2. Buffer (B) TSM- 20mM Tris-HCl, pH 7.4, 150mM NaCl, 2mM CaCl2, 2mM MgCl2
5.1.3. Buffer (C) TSM Binding Buffer (TSMBB) – TSM buffer + 0.05% Tween 20 + 1% BSA
5.1.4. dH2O
5.2. Prepare 400 µl of sample by diluting antibody in TSMBB or appropriate Binding Buffer based on properties of Antibody to a final concentration of 1-50 µg/ml, or an appropriate concentration required for the analysis.
5.2.1. Specific dilution information can be found in the Glycan Array Processing Form (GAPF). Always refer to this document for any exceptions needed for individual samples.
5.3. Remove slide(s) from the refrigerator, and add SecureSeal or Proplate chamber, avoiding printed array area(s).
5.4. Scan slide wet if previously used with MAWE protocol to ensure there is no residual binding before continuing
5.5. Hydrate the slide by adding 400ul to SecureSeal and set aside for 5 minutes.
5.6. Remove excess liquid from slide by aspiration, using the seal ports.
5.7. Add 400ul of diluted primary sample to the slide.
5.8. Incubate slide on orbital shaker, covered, and in the dark for 1 hr at RT.
5.9. After 1 hr incubation, aspirate sample off the slide.
5.10. Wash the slide by adding 400ul of Buffer A (TSMW) and removing by aspiration. Repeat 4x
5.10.1. Repeat Step 5.8 with Buffer B (TSM) 4x before the next step
5.11. Add 400 µl of AlexaFluor-488-labeled (or other label) secondary antibody.
5.12. Incubate as above (5.6)
5.13. Repeat washing steps after 1 hour incubation with secondary antibody (Steps 5.9 and 5.9.1)
5.14. Keep the fourth wash of TSM on the slide for scanning procedures.
5.15. Scan in a fluorescent Scanner at the appropriate wavelength (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 complete assay and performing MAWE as short as possible, this ensures the MAWE protocol will remove binding.
6.2. Place in a glass container and microwave for 3 seconds.
6.3. Aspirate SDS, and add fresh buffer. Repeat 10 times, replacing buffer between each microwave.
6.4. Wash the slide 2 more times with 10% SDS buffer, and then wash as in steps 5.10 and 5.10.1
6.5. Place slide into a slide tube with TSM Preservation Buffer
6.5.1. Store at 4°C 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 procedures on Glycan Array Processing Form (GAPF).
Record Slide number on GAPF and fill out worksheet log form regarding sample information and slide number.
***Some samples may require different buffers or conditions. Always refer to the GAPF for investigator details.***