Application

This kit can be used to measure the cysteine (cys) content in animal serum, plasma, tissue, cells and other samples. 

Detection significance

There are three kinds of sulfur-containing amino acids in protein: Methionine, Cystine and Cys. Cys is the only one sulfur amino acid which contains thiol. Cys is converted from methionine and can mutual transform with Cystine. Cys is involved in the formation of protein disulfide bonds, which usually acts as the component of protein activity center and can provide thiol for other physiological and biochemical reactions. In addition, Cys accumulated on the surface of skin and mucosal, and maintains the activity of important thiol enzyme in the formation of keratin. Cys also supplies the thiol to keep the normal metabolism of skin, and regulates the bottom melanin produced by the pigment cells in the lower epidermis, which has the function of whitening, detoxification, improve inflammation and allergies skin etc.

Detection principle

Phosphotungstic acid can be reduced by Cys and form tungsten blue, which has an absorption peak at 600 nm. Cys content can be calculated with the absorbance at 600 nm.

Experimental instruments

Spectrophotometer (600 nm), Refrigerated centrifuge, Micropipettor, 1 mL cuvette

Operation steps

1.     Extraction of Cys:

(1)  Extraction of Cys in liquid sample: take 0.1 mL of liquid sample, add 0.9 mL of Reagent 1 and mix fully. Centrifuge at 8000g for 10 min at 4℃, then take the supernatant for measurement.

(2)  Extraction of Cys in tissue sample: add the appropriate volume of Reagent 1 according to the ratio of Weight (g): Volume (mL) =1: 5~10 (It is recommended to weigh 0.1 g of tissue, and add 1 mL of Reagent 1). Mechanical homogenate the sample in ice water bath. Centrifuge at 8000 g for 10min at 4℃, then take the supernatant for measurement.

(3)  Extraction of Cys in bacteria or culture cells: collect the bacteria or culture cells into the centrifuge tube, centrifuge and discard the supernatant. Add Reagent 1 into the sediment according to the ratio of Bacteria or cells number: Reagent 1 (mL) =500~1000: 1(it is recommended to add 1 mL of Reagent 1 into 5×106 cells), then treat the sample with sonication on ice (power: 20% or 200W, 3 seconds/time, interval for 10 seconds, repeat 30 times). Centrifuge at 8000g for 10 min at 4℃. Take the supernatant and preserve it on ice for detection.

2.    Operation table:

(1)    Preheat the spectrophotometer for more than 30 min, set the wavelength at 600 nm and set to zero with distilled water.

(2)    Operation table

Note:

1. The kit is for scientific research only.

2. Instructions should be followed strictly, changes of operation may result in unreliable results.

3. The validity of kit is 3 months.

4. Do not use components from different batches of kit.

5. The minimum detection limit is 100 μmol/L.

6. If the Cys content is calculated by protein concentration, the protein concentration of the sample needs to be determined separately (E-BC-K318, E-BC-K168, E-BC-K165).