- ENZYME EXTRACTION:
1. Mill malt or barley to pass a 0.5 mm screen with a suitable laboratory mill (e.g. Buhler Miag disc mill DLFU; Setting fine, or Frisch Pulverisette 14® with 0.5 mm screen).
2. To exactly 0.5 g of flour in a 13 mL polypropylene tube (Sarstedt cat. no. 60.541.685 PP; www.sarstedt.com), add 5.0 mL of Buffer A (Bottle 2).
3. Allow the enzyme to extract over a 1 h period at room temperature, with frequent vigorous stirring on a vortex mixer (approx. 5 times over the 1 h period). Alternatively, place the tube into Stuart Blood Tube Rotator (http://design.hileytech.com/ fisher/Stuartblood.html) which allows end-over-end mixing and run the machine for 1 h.
4. Filter an aliquot of the enzyme preparation through Whatman GF/A glass fibre filter paper, or centrifuge in a bench or microcentrifuge at a minimum of 2,000 g for 10 min.
5. Add 0.2 mL of filtrate to 4.0 mL of Buffer B (bottle 3), mix and use this for the assay of β-amylase activity.
NOTE:
If the level of α-amylase is to be assayed in the same extract using the Ceralpha method, proceed as follows:
1. Add 0.2 mL of the diluted malt or barley extract (as used in the Betamyl-3® assay above ) to 3 mL of Ceralpha Buffer A (prepared as shown on page 3 of the Ceralpha Booklet K-CERA).
2. Proceed with assay of α-amylase (page 7 of this booklet).
- Assay of β-Amylase:
1. Dispense 0.2 mL aliquots of the diluted malt extract directly to the bottom of 13 mL polypropylene tubes and pre-incubate the tubes at 40°C for approx. 5 min.
2. Pre-incubate Betamyl-3® Substrate Solution at 40°C for approx. 5 min.
3. To each tube containing diluted malt extract add 0.2 mL of Betamyl-3® Substrate Solution, stir on a vortex mixer and incubate tubes at 40°C for exactly 10 min (from time of addition).
4. At the end of the 10 min incubation period, add 3.0 mL of Stopping Reagent and stir the tube contents.
5. Read the absorbance of the reaction solutions and the reagent blank at 400 nm against distilled water.
商品特色
商品規格
- 商品規格 100 檢測反應
- 此套組包含:
Bottle 1: *2
Each vial contains p-nitrophenyl-β-D-maltotrioside (PNPβ-G3) plus β-glucosidase (50 U) and stabilisers.
Stable for > 4 years below -10°C.
Bottle 2:
Tris/HCl buffer (Extraction Buffer) (25 mL, 1 M, pH 8.0) plus disodium EDTA (20 mM) and sodium azide (0.02% w/v).
Stable for approx. 4 years at 4°C.
Bottle 3:
MES buffer (Dilution Buffer) (48 mL, 1 M, pH 6.2) plus disodium EDTA (20 mM), BSA 10 mg/mL and sodium azide (0.09% w/v).
Stable for approx. 4 years at 4°C.
Bottle 4:
Cysteine hydrochloride (16 g).
Stable for > 4 years at room temperature.
Bottle 5:
Malt flour of standardised β-amylase activity (as specified on bottle label).
Stable for > 4 years; store sealed at room temperature.
