Amylase (AMS) Activity Assay Kit (Iodine Solution, Micro Method)

Cat. No.: A1515870

GRADE & PURITY BioReagent

Übersicht

Product Introduction 

Amylase (AMS), also known as 1,4-α-D-glucanohydrolase, refers to a general term for enzymes that hydrolyze starch and glycogen. Assays for amylase activity are mainly categorized into methods using native starch substrates and defined substrates. The former includes the iodine-starch method, while the latter comprises methods based on maltopentaose substrates and 4-NP-G substrates.

Amylase (AMS) Activity Assay Kit (Iodine Solution, Micro Method) : In this assay, α-amylase in samples such as serum or plasma catalyzes the hydrolysis of α‑1,4 glycosidic bonds in starch molecules to generate glucose, maltose, dextrins and other products; iodine solution combines with unhydrolyzed starch to form a blue complex, and the color intensity is compared with the blank without enzymatic reaction to calculate amylase activity units. Absorbance is read at 660 nm with a microplate reader. The kit is applicable for the determination of endogenous amylase activity in cell or tissue lysates/homogenates, plasma, serum, urine and other samples. This method is simple and easy to perform, requires no specialized equipment and features low reagent cost, and is widely used in China. Sample throughput will decrease significantly when a spectrophotometer is used. This kit is for research use only and not intended for clinical diagnosis or other purposes.  

Component

Component
Component No.	Component Name	Appearance	200 T	500 T	Storage	Quantity Per Test
A1515870A	AMS Assay Buffer	Colourless clear liquid	8 mL	20 mL	2-8℃	35 μL
A1515870B	KI Solution	Brown liquid	0.8 mL	2 mL	2-8℃. Store in the dark.	3.5 μL

Materials to be supplied by user:

1. Distilled water, normal saline

2. 96-well microplate

3. Microplate reader

Procedure (For reference only):

1. KI Working Solution: Bring KI Solution to room temperature. Mix KI Solution with distilled water at a ratio of 1:9 to prepare KI working solution. Store at 4 °C protected from light for up to 1 month.

2. Sample preparation: 

① Cell or tissue samples: Take appropriate cell or tissue lysate. Homogenize if necessary, perform low-speed centrifugation and collect the supernatant. Store at −20 °C for AMS assay. 

② Plasma, serum and urine samples: Prepare plasma and serum by routine protocols. Dilute 5–10 times with normal saline before assay. Urine samples are generally diluted 10–20 times with normal saline prior to measurement. Samples can be stored at −20 °C. A sample control containing sample but no substrate must be set to eliminate interference from intrinsic sample color. 

③ High-activity samples: Samples with high AMS activity may be diluted with normal saline or PBS.

④ After sample preparation, protein concentration (mg/mL) can be determined using BCA Protein Assay Kit for subsequent calculation of AMS content per unit mass of protein in tissues or cells.

3. AMS loading: Prepare a 96-well microplate and set up blank wells and test wells according to the table below. Add solutions sequentially and avoid bubble formation. If amylase activity in samples is excessively high, reduce sample volume or dilute the sample before testing. Assay samples in duplicate whenever possible.

Additive (μL)	Test Well	Control Well
AMS Assay Buffer	35	35
Incubate at 37 ℃ for 5 min.
Test Sample	7	-
Mix thoroughly, then incubate in a 37 ℃ water bath for exactly 7.5 min.
KI Working Solution	35	35
Distilled Water	210	217

4. AMS measurement: Mix gently, zero the microplate reader with distilled water, and measure the absorbance at 660 nm using a microplate reader. Record the absorbance values as Atest and Ablank.

Calculation of results  

Definition of amylase activity unit: One unit (U) of amylase activity is defined as the quantity of amylase in 100 mL serum that hydrolyzes 5 mg starch at 37 °C within 15 min. The AMS activity of samples was calculated based on this definition.

For plasma, serum and urine liquid samples: AMS activity (U/dl) = [(Ablank − Atest) / Ablank] × (0.4 × 0.035 × 15 × 100) / (5 × 7.5 × 0.007) × dilution factor = [(Ablank − Atest) / Ablank] × 80 × dilution factor

For cell or tissue samples: AMS activity (U/mg) = [(Ablank − Atest) / Ablank] × (0.4 × 0.035 × 15) / (5 × 7.5 × 0.007 × sample protein concentration) = [(Ablank − Atest) / Ablank] × 0.8 / sample protein concentration

Where: Ablank = absorbance of blank well; Atest = absorbance of test well; 0.4 = starch concentration (mg/mL) in AMS Assay Buffer; sample protein concentration = protein concentration of the tested sample.

Reference range (37 °C, healthy subjects)

Serum amylase activity: 80–180 U/dl 

Urine amylase activity: 100–1200 U/dl  

Notes

1. This kit can also be used with a spectrophotometer for detection, but the number of detectable samples will decrease sharply.

2. Oxalate, citrate, disodium EDTA and sodium fluoride inhibit AMS activity, whereas heparin has no inhibitory effect. Test samples must not contain AMS inhibitors, and repeated freeze-thaw cycles should be avoided.

3. Enzyme activity shows a linear relationship with substrate hydrolysis when the activity is below 400 U. If the absorbance of the test well is half that of the blank well, increase the sample dilution factor or reduce the loading volume of the test sample and repeat the measurement. The final result should be multiplied by the dilution factor.

4. This kit is also suitable for AMS detection of other sample types. Urine samples need to be diluted 10–20-fold before measurement.

5. Discard the AMS Assay Buffer if turbidity or flocculent precipitate appears.

6. For personal safety, wear a lab coat and disposable gloves during operation.

Appendix: Reaction curve of amylase concentration in mouse serum

1. Pretreatment: Mouse serum was diluted with normal saline at serial dilutions of 2-fold, 4-fold, 8-fold, 16-fold, 32-fold, 64-fold, 128-fold, 256-fold, 512-fold, 1024-fold, 2048-fold and 4096-fold. An aliquot of 7 μL of each diluted sample was taken for detection.

2. Operation table: (AMS Assay Buffer was pre-incubated at 37 °C for 5 min in advance)

Additive (μL)	Test Well	Control Well	Water Well
AMS Assay Buffer	35	35	-
Incubate at 37 ℃ for 5 min.
Test Sample	7	-	-
Mix thoroughly, then incubate in a 37 ℃ water bath for exactly 7.5 min.
KI Working Solution	35	35	-
Distilled Water	210	217	287

3.  Measurement results

Sample concentration (%)	Measured OD	Absolute OD (ΔOD)
Water control well	0.0779	–
Blank well	0.2281	0
100	0.07915	0.20895
50	0.08165	0.20645
25	0.08285	0.20525
12.5	0.08455	0.20355
6.25	0.08745	0.20065
3.125	0.1096	0.1785
1.5625	0.15355	0.13455
0.7813	0.19175	0.09635
0.3906	0.22575	0.06235
0.1953	0.24835	0.03975
0.0977	0.26495	0.02315
0.0488	0.2763	0.0118

4. Plot the curve as shown below

       

(As illustrated in the figure: the reaction curve is approximately linear when the absolute OD value ranges from 0 to 0.09635. Therefore, the sample concentration corresponding to this absolute OD range is the optimal sampling concentration.)

(The above curve is for preliminary test reference only.)

Specifications

Spezifikationen & Reinheit

BioReagent

Note

BioReagent

Anwendung

Biochemical assay, Cell Metabolism, Enzyme activity assay

Probentyp

Pflanzengewebe, Plasma, Serum, Tierische Gewebe, Urin

Methode des Nachweises

Micro Method

Quantitativ

Quantitative

Detection instrument

Microplate reader

Detection wavelength

660nm

Empfindlichkeit

Light-sensitive

Lagerung und Versand

Storage

Store at 2-8°C,Protected from light

Verschickt in

Wet ice

Stabilität und Lagerung

Each component has a shelf life of 1 year under corresponding storage conditions.

Contents & Storage

Component No.	Component Name	Appearance	200 T	500 T	Storage	Quantity Per Test
A1515870A	AMS Assay Buffer	Colourless clear liquid	8 mL	20 mL	2-8℃	35 μL
A1515870B	KI Solution	Brown liquid	0.8 mL	2 mL	2-8℃. Store in the dark.	3.5 μL

Häufig gestellte Fragen

What does BioReagent mean?

BioReagent indicates that the product has been processed and tested for biological and biochemical applications. Specifications may include endotoxin limits, microbial controls, sterility, low DNase/RNase/protease activity, and verified biological activity where relevant.

How should this product be stored?

Store at 2–8 °C, protected from light. The material is light-sensitive — keep it in its original opaque or amber container.

How is this product shipped?

This product ships chilled on wet ice. Unpack on arrival and transfer it to the storage condition stated above.

What documentation is provided?

Available product documentation, including Certificates of Analysis (COA), Safety Data Sheets (SDS), and specification sheets, is shown in the product document area. Document availability and access follow the current site policy.