Lysine assay kit

Product Number: LAK01

Shipping and Storage 

Store at 4℃ in dark for 12 months

Description

Lysine is one of the essential amino acids in the human body, which can promote human development, enhance immune function, and improve the function of central nervous system tissues. Lysine is an essential alkaline amino acid. Due to the low content of lysine in cereal foods and their susceptibility to damage and deficiency during processing, it is called the first limiting amino acid. This reagent kit provides a simple detection method that can detect the content of lysine in various biological samples. The principle is that lysine in proteins has a free εNH2 reacts with ninhydrin reagent to generate a blue purple substance with a characteristic absorption peak at 570nm; Calculate the lysine content by measuring the absorbance at 570nm.

Components

Component	48T	96T	Storage
Extraction solution	50mL	100mL	4℃
Buffer 1	6.25mL	12.5mL	4℃
Buffer 2	Powder	Powder	4℃, avoid light
Lysine standard	Powder	Powder	4℃, avoid light

Self provided consumables

1.Enzyme reader or visible spectrophotometer (capable of measuring absorbance at 570nm) and water bath

2.96 well plate or trace glass colorimetric dish, adjustable pipette and nozzle

3.Low temperature centrifuge

4.Deionized water, ethanol

5.Homogenizer (if it is a tissue sample)

Reagent preparation

Note: Before opening the cap of each component (small tube reagent), please centrifuge at low speed first.

1.Extraction solution: Ready to use; Before use, balance to room temperature; Store at 4 ℃.

2.Buffer 1: Ready to use type; Before use, balance to room temperature; Store at 4 ℃.

3.Buffer 2: Prepare before use. Dissolve 96T in 12.5mL of 95% ethanol and 48T in 6.25mL of 95% ethanol. Store unused and dissolved Buffer 2 at 4℃ in the dark for one week. If long-term storage is required, please store at 20℃ after packaging to avoid repeated freeze-thaw cycles.

4.Preparation of working solution: Prepare according to the required sample size in a ratio of Buffer 1: Dissolved Buffer 2=1:1.

5.Lysine standard: Prepare before use, add 1.71mL of deionized water, fully dissolve to obtain 40μmol/mL standard. Unused dissolved standard can be stored at 4 ℃ in the dark for one week. If long-term storage is required, please store at 20℃ after packaging to avoid repeated freeze-thaw cycles.

6.Standard curve setting: Dilute the 40μmol/mL standard with deionized water as shown in the table below.

	Standard volume	Extraction solution volume(µL)	Standard concentration(μmol/mL)
Std.1	20µL of 40μmol/mL	380	2
Std.2	200µL of Std.1(2μmol/mL)	200	1
Std.3	200µL of Std.2 (1μmol/mL)	200	0.5
Std.4	200µL of Std.3 (0.5μmol/mL)	200	0.25
Std.5	200µL of Std.4 (0.25μmol/mL)	200	0.125
Std.6	200µL of Std.5 (0.125μmol/mL)	200	0.0625
Std.7	200µL of Std.6 (0.0625μmol/mL)	200	0.0313

Note: Please use newly prepared standard samples for each experiment.

Sample preparation

1.Animal tissue:Weigh approximately 0.1g of the sample, add 1mL of Extraction solution, homogenize thoroughly at room temperature, transfer to a 1.5mL EP tube, cover tightly (to prevent water loss), and extract in an 80 ℃ water bath for 20 minutes; After cooling, centrifuge 10000g at room temperature for 10 minutes, and take the supernatant for testing.

2.Plant tissue:Weigh approximately 0.1g of the sample, add 1mL of Extraction solution and crush. Crush at room temperature with ultrasound for 5 minutes (power of 20% or 200W, ultrasound for 3 seconds, interval of 7 seconds, repeat 30 times), transfer to a 1.5mL EP tube, cover tightly (to prevent water loss), and extract in an 80 ℃ water bath for 20 minutes; After cooling, centrifuge 10000g at room temperature for 10 minutes, and take the supernatant for testing.

3.Cells or bacteria:Collect 5 million cells or bacteria into a centrifuge tube, wash the cells with cold PBS, discard the supernatant after centrifugation, add 1mL Extraction solution, crush at room temperature with ultrasound for 5 minutes (power of 20% or 200W, ultrasound for 3 seconds, interval of 7 seconds, repeat 30 times), transfer to a 1.5mL EP tube, cover tightly (to prevent water loss), and extract in an 80 ℃ water bath for 20 minutes; After cooling, centrifuge 10000g at room temperature for 10 minutes, and take the supernatant for testing.

4.Cell supernatant or serum (plasma): In a 1.5mL EP tube, take 0.5mL of liquid and add 0.5mL of extraction solution. Cover tightly (to prevent water loss) and place in an 80 ℃ water bath for extraction for 20 minutes; After cooling, centrifuge 10000g at room temperature for 10 minutes, and take the supernatant for testing.

Note: It is recommended to use fresh samples. If the experiment is not conducted immediately, the samples can be stored at 80℃ for 6 months. It is recommended to use the BCA protein quantification kit for sample protein concentration determination.

---