F. keratolyticus Endo-beta-Galactosidase Protein, CF

• Labeling Buffer: 25 mM MES, 0.5% (v/v) Triton® X-100, 2.5 mM MgCl₂, 2.5 mM MnCl₂, 1.25 mM CaCl₂, 0.75 mg/mL BSA, pH 7.0
• Assay Buffer: 0.1 M MES, pH 6.0
• Gel Running Buffer: 40 mM Tris, 1 mM EDTA, adjust to pH 8.0 with acetic acid
• Recombinant F. keratoyticus Endo-beta -Galactosidase (rF.k. Endo-beta -galactosidase) (Catalog # 8620-GH)
• Goat Keratan Sulfate Proteoglycans (Catalog # 8618-KS)
• Recombinant Human Carbohydrate Sulfotransferase 1/CHST1 (rhCHST1) (Catalog # 5316-ST)
• 8% SDS-PAGE (approximately 15 cm x 20 cm, 20 lanes per gel)
• PAP³⁵S (prepared in-house using the PAPS Synthesis Kit (Catalog # EA005), ~1 μM  = ~2 x 10⁶ cpm/μL)
• Gel loading buffer: 0.15 M Tris, 20.8 mM SDS, 1.15 M Glycine, 174 µM Bromophenol Blue, 30% Glycerol 
• Blotting paper (Fisher Scientific, Catalog # 05-714-4)
• Gel dryer
• Glogos® II autorad markers (Stratagene, Catalog # 420202) or equivalent
• Blue sensitive medical X-ray film
• X-ray film cassette
• Film developer (Konica SRX-101A Medical Film Processor) or equivalent
• Liquid scintillation counter (Beckman Coulter, Model # LS5000TD) or equivalent
• Liquid scintillation fluid (Beckman Coulter, Catalog # 141349) or equivalent

1. Create Radiolabeled Keratan Sulfate Mixture containing 0.1 mg/mL Keratan Sulfate, 12 μg/mL rhCHST- 1, and 0.025 μM PAP³⁵S in Labeling Buffer.
2. Incubate Keratan Sulfate Mixture at 37 °C for 1.5 hours.
3. Dilute incubated Keratan Sulfate Mixture 3 fold in Assay Buffer.
4. Dilute rF.k. endo-beta -galactosidase to 2.667, 0.667, 0.2667, 0.133, 0.0667, 0.02, 0.00667, and 0.00133 µg/mL in Assay Buffer.
5. Combine 15 µL of rF.k. endo-beta -galactosidase at each dilution with 15 µL diluted Keratan Sulfate Mixture. Include a control containing 15 µL Assay Buffer and 15 µL Keratan Sulfate Mixture.
6. Incubate all reactions at 37 °C for 20 minutes.
7. Add 15 µL gel loading buffer to each reaction. Mix.
8. Load 30 µL of each reaction per lane on a gel. Leave empty lanes between samples.
9. Run at 200 V for 30 minutes.
10. Transfer gel onto blotting paper and dry with gel dryer for 1 hour or until fully dry.
11. Affix two autorad markers to the blotting paper next to the dried gel.
12. In a darkroom expose dried gel to X-ray film by enclosing overnight in a cassette. Develop the following day.
13. Using the dried gel, begin marking regions to be cut out for scintillation counting.
14. Mark a horizontal line across the top of the entire gel just under the bottom of the wells.
15. Using the developed film as an overlay, mark a second line just below the lower edge of the labeled Keratan Sulfate for each well.
16. Draw a third line just below where the labeled product migrated (ignore any free sulfate, appearing equivalent in all lanes, and migrating the furthest). For the control, identify the empty region where the product would appear.
17. The area between the first two lines is considered to contain the labeled starting material. The area between the second two lines is considered to contain the compact cleavage product resulting from the reaction.
18. Mark vertical lines distinguishing one lane (reaction condition) from another.
19. Cut each region (two per lane) and place each into a separate liquid scintillation vial. Add 5 mL liquid scintillation fluid to each vial and count the vials for ³⁵S.
20. Determine the amount of rF.k. Endo-beta -galactosidase required for 50% cleavage by plotting % cleavage vs. ng of
    rF.k. Endo-beta -galactosidase with 4-PL fitting.
    
    

Per Reaction:

• Keratan Sulfate: 0.5 μg
• rF.k. Endo-beta -galactosidase: 40, 10, 4, 2, 1, 0.3, 0.1 and 0.02 ng