Sample preparation method for measuring sex hormone level of Japanese flounder larvae

A technique for sample preparation and flounder larvae, which is applied in the field of sample preparation in the determination of sex hormones, achieves the effects of wide application range, reduced physical injury, and stable results

Inactive Publication Date: 2009-01-14
INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no reports on the determination and regulation of sex hormone levels in juvenile marine fish larvae in China, and there are only reports from American scholars abroad that the extraction of flounder with ether and freezing and separation of methanol can improve the sex hormone levels of juvenile flounder. Determination and Analysis of Alcohol Hormone Level

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] 1) Take fresh juvenile flounder (full length, 0.8cm), weigh (0.004g), wash the whole fish twice with distilled water, and place it in a mortar.

[0023] 2) Add a small amount of PBS buffer (containing 137mmol / L NaCl, 2.7mmol / L KCl, 10mmol / LNa 2 HPO 4 and 2mmol / L KH 2 PO 4 , pH 7.4) until soaked, fully ground with liquid nitrogen, and transferred the homogenate into a centrifuge tube; added PBS buffer solution to 10 times the fish body weight value (this time it should be filled to 1mL), and then ultrasonically crushed for 20 seconds bell.

[0024] 3) Diethyl ether in an amount twice the volume of PBS buffer solution was added to the homogenate, mixed by inverting for 5 minutes, and then centrifuged at 6000 rpm for 10 minutes at 4°C.

[0025] 4) Collect the liquid phase, add 6 times the volume of PBS buffer to the solid phase, then add ether with 1 times the volume of the PBS buffer, mix well, and centrifuge at 6000 rpm for 10 minutes at 4°C; collect the liquid phase...

Embodiment 2

[0031] 1) Take fresh juvenile flounder (full length, 3.2cm), weigh (0.55g), wash the whole fish 3 times with distilled water, cut it into pieces with scissors, and place it in a mortar.

[0032] 2) Add a small amount of PBS buffer solution (containing 137mmol / L NaCl, 2.7mmol / L KCl, 10mmol / L NaCl, 2 HPO 4 and 2mmol / L KH 2 PO 4 , pH 7.4) until soaked, fully ground with liquid nitrogen, and the homogenate was transferred to a centrifuge tube; then continue to add PBS buffer solution, repeat the operation, and finally add PBS buffer solution to 9 times the value of the fish body weight, and then use ultrasonic pulverization 30 seconds.

[0033] 3) Diethyl ether in an amount twice the volume of PBS buffer solution was added to the homogenate, mixed by inverting for 5 minutes, and then centrifuged at 7000 rpm for 9 minutes at 4°C.

[0034] 4) Collect the liquid phase, add 5 times the volume of PBS buffer to the solid phase, then add ether 1 times the volume of the PBS buffer, mi...

Embodiment 3

[0040] 1) Take juvenile flounder (full length, 6.5 cm) stored at -80°C, weigh (2.39 g), wash the whole fish 3 times with distilled water, cut it into pieces with scissors, and place it in a mortar.

[0041] 2) Add a small amount of PBS buffer solution (containing 137mmol / L NaCl, 2.7mmol / L KCl, 10mmol / L NaCl, 2 HPO 4 and 2mmol / L KH 2 PO 4 , pH 7.4) until soaked, fully ground with liquid nitrogen, and the homogenate was transferred to a centrifuge tube; then continue to add PBS buffer solution, repeat the operation, and ensure that the final amount of PBS buffer solution added is 8 times the value of the fish body weight, Ultrasonic pulverization was then performed for 40 seconds.

[0042] 3) Add ether twice the volume of PBS buffer solution to the homogenate, invert and mix for 5 minutes, and then centrifuge at 8000 rpm for 8 minutes at 4°C.

[0043] 4) Collect the liquid phase, add 4 times the volume of PBS buffer solution to the solid phase, then add ether with 1 times th...

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PUM

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Abstract

The invention discloses a sample preparation method for the sex hormone level determination of larval paralichthyidae fishes to young paralichthyidae fishes and comprises the following steps: a larval paralichthyidae fish or a young paralichthyidae fish is prepared, measured, skived after adding liquid nitrogen, ultrasonically grinded and evenly mixed with ethyl ether; a liquid phase is stored after centrifugation, a solid phase is evenly mixed with PBS buffering liquid and ethyl ether, and the liquid phase is stored after centrifugation; the liquid phase is evenly mixed, statically placed and delaminated; the delaminated liquid is placed into liquid nitrogen for being quickly cooled, and an organic phase is recycled; an aqueous phase is mixed with ethyl ether, statically placed, delaminated and quickly cooled in liquid nitrogen, and the organic phase is recycled, collected and placed in a stink cupboard, and then volatilized in a water bath at the temperature of 45 to 55 DEG C; the organic phase is dissolved in the PBS buffering liquid, stored at the temperature of minus 20 DEG C, and prepared for radioimmunoassay. The sample preparation method has simple operation process and stable result, can be used for the sample preparation of the sex hormone content determination of the larval paralichthyidae fishes to the young paralichthyidae fishes with the length of 6.5 cm, and is applicable to the internal secretin regulation and control research of other young flounder fishes even other marine fishes, thus having wide application range.

Description

technical field [0001] The invention relates to a sample preparation technology in the determination of sex hormones, in particular to a sample preparation method in the determination of sex hormone levels in juvenile flounder fish, an important marine culture object in my country. Background technique [0002] The determination of sex hormones has been used in various research and testing. In fish, by detecting the level of sex hormones in fish, it is possible to study the growth and development of fish, especially the development of sexual organs and reproductive cycle; there are also the effects of exogenous sex hormones on fish itself, as well as fish sex differentiation and sex reversal, etc. . In addition, this technology can also be applied to the extraction and measurement of other types of hormones. [0003] At present, the level of sex hormones is mainly obtained by measuring the content in serum samples, and it is almost difficult or even impossible to obtain th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28
Inventor 孙鹏尤锋杨奔张培军徐永立
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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