1. Introduction:
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. Removal of binding with the MAWE method, for future reuse of the slide.
2. References:
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 etched bar code - DO NOT TOUCH THIS AREA
3.2. SecureSeal Seals (Grace Biolabs, 621506) or ProPlate Chambers (Grace Biolabs, 246868)
3.3. Orbital Shaker (Belco Biotechnology)
3.4. Aspirator
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. dH20
3.10. Bovine Serum Albumin (BSA), Protease Free, LY-0081 (Boval Company)
3.11. Cy5-Streptavidin (Invitrogen, 434316)
3.12. Tween-20 (EMD Biosciences, 655205)
3.13. Sodium Azide (Fisher scientific, S227-500)
3.14. Glycerol (Sigma, G5516-500)
3.15. Sodium Dodecyl Sulfate (Sigma-Aldrich, L3771-100)
3.16. Pico ImageXpress (Molecular Devices, LLC) or similar scanner capable of holding the slide with the SecureSeal or ProPlate chambers.
3.17. Biotin-Tagged samples
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 (SDS)= 10% SDS
Comment: For buffer preparation, see Direct Glycan Binding Assay protocol: 4. Buffers
5. Assay 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- 20mM Tris-HCl, pH 7.4 150mM NaCl, 2mM CaCl2, 2mM MgCl2
5.1.2. Buffer (B)- TSM Wash Buffer (TSMW) - TSM Buffer + 0.05% Tween-20
5.1.3. Buffer (C)- TSM Binding Buffer (TSMBB) – TSM buffer +0.05% Tween 20 + 1% BSA
5.1.4. Buffer (D)- TSM Preservation Buffer- TSM Buffer + 0.05% Tween-20 + 0.2% Sodium Azide + 5% Glycerol
5.1.5. Buffer (E)- 10% Sodium Dodecyl Sulfate (SDS)
5.1.6. dH2O
5.2. Remove slide(s) from desiccator and place the SecureSeal or ProPlate onto the slide. Be sure to seal around the scan area.
5.3. Hydrate the slide by adding 400 µL of ‘Buffer A’ (usually TSMW) for 5 minutes.
5.3.1. If this slide was used previously, scan this slide before continuing to verify the previous binding was removed.
5.4. Sample Preparation: Prepare 400 µl of sample by diluting biotin-tagged 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.
5.5. Aspirate hydration liquid from the slide using a bench vacuum aspirator
5.6. Add 400 µl of sample (see 5.4) to the slide using the port of the SecureSeal or directly into the ProPlate wells, without touching the slide.
5.7. Incubate slide on the orbital shaker covered for 1 hour at RT.
5.8. After 1 hour incubation, remove sample solution using the aspirator.
5.9. Wash the slide by adding 400 µL of ‘Buffer A’ and aspirating 4 times (TSMW).
5.10. Repeat this process with 4 washes of 400 µL of ‘Buffer B’ (TSM).
5.11. Prepare 400 µL of Streptavidin-Cy5 solution, diluted to working concentration, as required (ex. 5 µg/ml).
5.12. Add 400 µl of Streptavidin-Cy5 to slide as above (5.6, 5.7) and incubate as above (5.8).
5.13. After 1 hour incubation, remove detection solution using the aspirator.
5.14. Wash the slide by repeating the same process in steps 5.9 and 5.10, making sure to not aspirate the final wash of ‘Buffer B’, TSM.
5.15. Scan under wet conditions on the scanner at 488 nm and 635 nm. (See Scanning Protocol).
6. MAWE Protocol:
6.1 Wash slide 2 times with 10% SDS, then add 1,000ul of SDS buffer to the slide in the SecureSeal or ProPlate (ProPlate can take more volume).
6.2 Place the slides individually on a petri dish and microwave for 3 seconds.
6.3 Aspirate SDS off the slide, then replace with fresh 10% SDS, and microwave as described above.
6.3.1 Repeat this process for a total of 10 times.
6.4 Wash 2x with 10% SDS buffer, then 4 times each of TSMW and TSM to eliminate any residual SDS.
6.5 If during the microwave process, the SecureSeal comes off, spin the slide dry, and add a new seal.
6.6 Rehydrate the slide with TSMW buffer, and scan the slide to check for removal of binding.
6.7 Perform a scan to make sure it is clean. If it is not clean, try repeating the MAWE process.
7. Protocol Notes
7.1 Record all procedures and any modifications in the Glycan Array Processing Form (GAPF)
7.1.1 Be sure to record the slide number in the GAPF
7.1.2 Enter slide type, number, and sample information into the logbook on the Lab shared server
7.2 Some samples may require different buffers than stated above. Always consult with investigator provided details from the GAPF.