1. Introduction:
1.1. Primary objective is to determine the binding specificity of Glycan Binding Proteins (GBPs) submitted by investigators. The primary array platform is the CFG printed defined glycan microarray.
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. Aspirator
3.3. Orbital Shaker
3.4. SecureSeal or ProPlate chambers (Grace Biolabs, 621506)
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. BSA (Fisher scientific, Bp1600-100) or Boval Bovine albumin, protease free (LY-0081)
3.11. Tween-20 (EMD Biosciences, 655205)
3.12. Glycerol (ThermoFisher, G5516)
3.13. ImageXpress Pico (Molecular Devices, LLC) or similar scanner
3.14. Unlabeled sample (protein, organism) (could have a tag/fusion protein)
3.15. Unlabeled primary antibody towards the unlabeled sample (could be towards the tag/fusion protein on the sample)
3.16. Fluorescently labeled secondary antibody to unlabeled primary antibody (correct species, type, isotype specificity)
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
NOTE: For specific buffer preparation instructions, see “Direct Binding Assay + MAWE” Document
5. Protocol:
5.1. Take out reagents and bring to RT
5.1.1. Buffer (A) TSM
5.1.2. Buffer (B) TSMW
5.1.3. Buffer (C) TSMBB
5.1.4. Buffer (D) TSMPB
5.1.5. dH2O
5.2. Remove slides from fridge, wash with water, and spin dry in a slide centrifuge.
5.3. Rehydrate slide with 400uL of TSMW buffer for at least 5 minutes
5.4. Scan the slide wet to ensure that there is no residual binding.
5.5. Prepare 400 µl of sample by diluting in TSMBB or appropriate Binding Buffer based on properties of sample, and the target testing concentration
5.5.1. Note: If an unlabeled polyclonal is used in the second step to detect the GBP, it should be tested first by itself to determine if it binds any glycans. Test the antibody at the same dilution as will be used with the sample.
5.6. Aspirate the TSMW from the slide.
5.7. Add 400uL of diluted sample to the slide
5.8. Incubate the slide, covered, on the orbital shaker for 1 hour.
5.9. Aspirate the sample from the slide
5.10. Wash the slide 4x with TSMW buffer
5.10.1. Repeat 4x with TSM buffer.
5.11. Add 400 µL of appropriate primary antibody (unlabeled monoclonal or polyclonal antibody or other unlabeled detecting reagent) to the slide
5.12. Incubate as in step 5.8
5.13. Repeat washing steps 5.10 and 5.10.1
5.14. Add 400 µl of appropriate fluorescently labeled secondary antibody.
5.15. Incubate as in step 5.8.
5.16. Repeat washing steps 5.10 and 5.10.1
5.16.1. Leave the 4th wash of TSM on the slide
5.17. Scan at the appropriate wavelength for the labeled secondary (See Scanning Protocol).
6. MAWE Protocol
6.1. After scanning, aspirate off the buffer and add 400 µl of 10% SDS
6.1.1. Wash 2x with 10% SDS, aspirating off after each wash
6.2. Microwave for 3s in a glass container
6.3. After microwaving, aspirate off the remaining SDS and add 1000 µl of fresh 10% SDS
6.3.1. Microwave 10x with 10% SDS, aspirating off after each wash and adding fresh SDS
6.4. After microwaving rounds, aspirate off the remaining buffer and add 1000 µl of 10% SDS
6.4.1. Wash 2x with 10% SDS, aspirating off after each wash
6.4.2. Wash 4x with TSMW, aspirating off after each wash
6.4.3. Wash 4x with TSM, aspirating off after each wash
6.5. Wash with water, and store in a slide tube with TSM PB
6.5.1. Store at 4C until next use.
6.6. If using the slide for another assay immediately, 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. Be sure to note any specific information on the unlabeled primary/detecting antibody and note if the primary antibody was tested alone.
***Some samples need special conditions or buffers. Always refer to the GAPF for investigator provided notes.***