1. Introduction
1.1 Primary objective is to determine the binding specificity of tagged Glycan Binding Proteins (GBPs) submitted by investigators. The primary array platform is the CFG printed defined glycan microarray. Subsequently, remove the binding using the MAWE protocol 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, glycans 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 Hybridization Chamber (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 diH2O
3.10 BSA (Fisher scientific, Bp1600-100) or Boval Bovine albumin, protease free (LY-0081)
3.11 Tween-20 (EMD Biosciences, 655205)
3.12 Sodium Dodecyl Sulfate (Sigma-Aldrich, L3771-100)
3.13 Sodium Azide (Fisher scientific, s227-500)
3.14 Glycerol (Sigma, G5516-500)
3.15 Pico ImageXpress (Molecular Devices, LLC) or similar scanner
3.16 Tagged/labeled sample (ex. His tag, Fc fusion, etc.)
3.17 Secondary Antibody to tag/label
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, see Direct Binding Protocol.
5. Assay Protocol
5.1 Remove the slide(s) from the fridge, wash with water, and spin dry in the slide centrifuge.
5.2 Attach the SecureSeal to the slide.
5.3 Rehydrate slides by adding 400 µl of TSMW and incubate for at least 5 minutes.
5.4 Scan the slide wet to check for any residual binding from previous assays.
5.5 Sample Preparation:
5.5.1 Prepare at least 400 µl of sample by diluting the labeled Glycan Binding Protein (GBP) or Organism in TSMBB or appropriate Binding Buffer based on properties of GBP or Organism to an appropriate final concentration required for the analysis.
5.6 Aspirate off TSMW.
5.7 Add 400µl of sample to subarray.
5.8 Shake for one hour (shaker set to speed 3) with slide covered.
5.9 After 1 hour, aspirate off sample and add 400 µl of TSMW.
5.9.1 Wash x 4 with TSMW, aspirating off after each wash.
5.9.2 Wash x 4 with TSM, aspirating off after each wash.
5.10 Add at least 400 µl of fluorescent secondary to subarray.
5.10.1 635nm wavelength is preferred for detection.
5.11 Shake for one hour (shaker set to speed 3) with slide covered (sample is fluorescent).
5.12 After 1 hour, aspirate off sample and add 400 µl of TSMW.
5.12.1 Wash x 4 with TSMW, aspirating off after each wash.
5.12.2 Wash x 4 with TSM, aspirating off after each wash.
5.13 Scan slide wet using scanning parameters for appropriate wavelength for the labeled scanner (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 1x more 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 TSMPB.
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.
*Keep in mind that the landing lights on the CFG slides are printed ONLY in 488
***Some samples may use different buffers or conditions. Check the Glycan Array Processing Form (GAPF) for investigator notes.***