Pepsin Activity Assay Kit

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Pepsin Activity Assay Kit

SKU #	E-BC-K844-M
Detection Instrument	Microplate reader (650-670 nm, optimum wavelength: 660 nm)
Detection method:	Colorimetric method

Product Details

Properties

Sample type	Gastric, intestinal tissue
Sensitivity	0.63 U/mL
Detection range	0.63-94.98 U/mL
Detection method	Colorimetric method
Assay type	Enzyme Activity
Precision	Average inter-assay CV: 4.8-5.6% \| Average intra-assay CV: 1.3-2.2%
Storage	-20℃
Valid period	12 months

Dilution of Sample

The recommended dilution factor for different samples is as follows (for reference only):

Sample type	Dilution factor
20% Rat gastric tissue homogenate	1
20% Mouse gastric tissue homogenate	1
20% Rat intestinal tissue homogenate	1
20% Mouse intestinal tissue homogenate	1

Note: The diluent is buffer solution. For the dilution of other sample types, please do pretest to confirm the dilution factor.

Detection Principle

Pepsin can catalyze substrate hydrolysis, and the hydrolysate reacts with the chromogenic agent under alkaline conditions to form a blue substance. The blue substance has a maximum absorption peak at 660 nm, and the color depth is proportional to the activity of pepsin. The enzyme activity can be calculated by OD value

Kit Components & Storage

Item	Component	Size (96 T)	Storage
Reagent 1	Buffer Solution	55 mL × 2 vials	-20℃, 12 months
Reagent 2	Precipitating Agent	50 mL × 1 vial	-20℃, 12 months
Reagent 3	Chromogenic Agent A	20 mL × 1 vial	-20℃, 12 months
Reagent 4	Chromogenic Agent B	10 mL × 1 vial	-20℃, 12 months, shading light
Reagent 5	Substrate	Power × 2 vials	-20℃, 12 months, shading light
Reagent 6	Substrate Solvent	12 mL × 1 vial	-20℃, 12 months
Reagent 7	1 mg/mL Standard Solution	1.5 mL × 1 vial	-20℃, 12 months, shading light
	Microplate	96 wells	No requirement
	Plate Sealer	2 pieces

Note: The reagents must be stored strictly according to the preservation conditions in the above table. The reagents in different kits cannot be mixed with each other. For a small volume of reagents, please centrifuge before use, so as not to obtain sufficient amount of reagents.

Technical Data:

Parameter:

Intra-assay Precision

Three rat gastric tissue samples were assayed in replicates of 20 to determine precision within an assay (CV = Coefficient of Variation).

Parameters	Sample 1	Sample 2	Sample 3
Mean (U/mL)	3.80	46.80	73.50
%CV	2.2	1.6	1.3

Inter-assay Precision

Three rat gastric tissue samples were assayed 20 times in duplicate by three operators to determine precision between assays.

Parameters	Sample 1	Sample 2	Sample 3
Mean (U/mL)	3.80	46.80	73.50
%CV	4.8	5.6	5.2

Recovery

Take three samples of high concentration, middle concentration and low concentration to test the samples of each concentration for 6 times parallelly to get the average recovery rate of 95%.

	Standard 1	Standard 2	Standard 3
Expected Conc. (μg/mL)	15	32	45.5
Observed Conc. (μg/mL)	14.7	29.8	42.8
Recovery rate(%)	98	93	94

Sensitivity

The analytical sensitivity of the assay is 0.63 U/mL. This was determined by adding two standard deviations to the mean O.D. obtained when the zero standard was assayed 20 times, and calculating the corresponding concentration.

Standard Curve

As the OD value of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique or temperature effects), so the standard curve and data are provided as below for reference only:

Concentration (μg/mL)	0	12	18	24	36	42	48	60
OD Value	0.041	0.163	0.222	0.285	0.411	0.476	0.53	0.647
OD Value	0.041	0.164	0.226	0.286	0.408	0.469	0.529	0.648
Average OD	0.041	0.164	0.224	0.286	0.410	0.473	0.530	0.648
Absoluted OD	0	0.123	0.183	0.245	0.369	0.432	0.489	0.647