Search within:

Hydrolytic Enzyme Analysis

By Jared L. DeForest

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

View Site in Mobile | Classic
Share by: