Hydrolytic Enzyme Analysis
Buffer
Acetate buffer (Stock Solution; 1 M)
In 500 ml Beaker
- Fill two-thirds with DI water.
- 68.304 g Sodium acetate trihydrate
Using a stir bar, bring to pH 5.5 by adding, with a pasture pipette, concentrated HCl.
Transfer to 500 ml volumetric flask and bring to volume with DI water.
Store in an opaque bottle at 4°C and use within 7 days.
Acetate buffer (Working Solution; 50 mM)
In 2 L volumetric flask
- 100 ml Acetate buffer stock solution
- Bring to volume with DI water.
Two liters is enough for 15 samples. Put in a carboy w/spout for easy dispensing.
Standards
Methylumbelliferyl (MUB; 10 µM)
In a 500 ml opaque bottle
- 500 g DI water (i.e, 500 ml)
- 0.881 mg 4-Methylumbelliferone
Mix very well.
Amino-Methylcoumarin (AMC; 10 µM)
In a 500 ml opaque bottle
- 500 g DI water (i.e, 500 ml)
- 0.876 mg 7-amino-4-methylcoumarin
Mix very well
Substrates (200 µM)
In a 50 ml opaque bottle
Weigh on a microbalance in tin capsules.
[NOTE: Please put the following in as a photo, not a table.]
Substrates | Enzyme | ID | mg / 50 ml Assay (hours)
- 4-MUB β-D-glucopyranoside | β-1,4-glucosidase | BG | 3.383 | 3 to 6
- 4-MUB beta-D-cellobioside | β-D-1,4-cellobiosidase | BC | 5.005 | 3 to 6
- 4-MUB N-acetyl-β-D-glucosaminide | β-N-acetylglucosaminidase | NAG | 3.794 | 0.5 to 3
- 4-MUB phosphate | Phosphomonoesterase | PM | 2.562 | 0.5 to 3
- bis-(4-MUB) phosphate | Phosphodiesterase | PD | 4.143 | 3 to 6
- L-Leucine-7-AMC | Leucine amino peptidase | LAP | 3.248 | 6 to 24
Add the correct about of DI water to achieve correct concentration, gravimetrically. Mix well and store at 4°C. Discard after 3 days. Assay each enzyme at least four times and at least 30 minutes apart for PM & NAG or 1 hour apart for BG, BC, PD, and LAP.
Created: 08/19/2010 Revised: 07/09/12