Figure 1. Representative diagram of substrate and product migration.
Dilute recombinant mouse CHST5 (R&D Systems, Catalog # 5210-ST) to 20 µg/mL, 10 µg/mL, 5 µg/mL, and 2.5 µg/mL in Assay Buffer 1. Dilute Methyl 6-O-(N-acetyl- Methyl 6-O-(N-acetyl-b-D-glucosaminyl)-a-D-mannopyranoside (abbreviated as GlcNAcMan; Sigma, Catalog # M0774), to make a 20 mM stock solution in deionized water.
Combine 15 µL 1 mM PAPS, 15 µL 35 S-PAPS, 15 µL 20 mM GlcNAcMan, and 135 µL Assay Buffer 1 (sufficient for ~9 reactions).
Start the reactions by combining 15 µL of enzyme at each dilution with 15 µL of the reaction mixture.
Include a 0 ng control containing 15 µL of the assay buffer and 15 µL of the reaction mixture.
Incubate at 37° C for 20 minutes.
Add 30 µmL 2X SDS gel loading buffer to each reaction (stops reaction and prepares for electrophoresis). Mix.
Load 20 mL of each reaction onto a gel. Leave empty space between each sample loaded.
Run electrophoresis at 200 volts for 30 minutes. Note: The loading buffer should run no more than halfway into the gel.
Transfer the gel onto blotting paper and dry on a gel dryer until completely dry.
Affix two autorad markers to the blotting paper containing the dried gel.
In a darkroom, expose the blotting paper with the dried gel to X-ray film. Place in a film cassette and expose overnight.
Develop the film.
Align the autorad markers on the film with the markers on the blot.
Determine where the assay product, the unreacted PAPS, and the free sulfate (if any) migrated on the gel (Figure 1). For the 0 ng control, identify the empty region where the product would have migrated if any had been produced.
Cut out the regions in each lane of the gel that contain the product and the unreacted PAPS.
Place each excised region into a separate liquid scintillation vial.
Add 5 mL liquid scintillation fluid to each liquid scintillation vial.
Use a liquid scintillation counter to count the amount of 35 S in each vial.
Enzyme Activity calculation:
Activity (pmolse/min) =
Where: S = applied donor substrate (pmoles) = 1,250 pmoles in this protocol
Ci = incorporated radioisotope (counts from product)
Ct = total applied radioisotope (counts from product and unreacted PAPS)
t = reaction time (min)
Plot activity vs. enzyme (µg) for specific activity. Specific activity = slope of the line.
Final Assay Conditions/reaction
PAPS: 1,250 pmoles (the amount of 35 S-PAPS in the assay is negligible)
rmCHST5: 0.3 µg, 0.15 µg, 0.075 µg, 0.0375 µg, 0 µg
GlcNAcMan: 25,000 pmoles