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Detection kit and detection method of phosphorylation of sperm tyrosine

A technology of tyrosine phosphorylation and detection kits, which is applied in biological testing, material inspection products, etc., can solve the problems of insufficient detection reagents, scarce sources of human oocytes, complicated follow-up operations of hamsters, etc., to reduce operations Step and operation time, good repeatability, effect of reducing detection cost

Active Publication Date: 2013-09-04
BRED LIFE SCI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These assays can use oocytes stored in saline, but assays are limited by the scarcity of human oocyte sources
[0003] The existing technology uses hamster eggs instead of human oocytes as a screening method. This method has the following disadvantages: ① hamster breeding is cumbersome; ② hamster egg retrieval and follow-up operations are complicated; ③ detection scheme reagents are not perfect; ④ detection cost is expensive

Method used

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  • Detection kit and detection method of phosphorylation of sperm tyrosine
  • Detection kit and detection method of phosphorylation of sperm tyrosine
  • Detection kit and detection method of phosphorylation of sperm tyrosine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1. Kit preparation

[0058] 1. Preparation of capacitation buffer

[0059] 1) Weigh 4.766g of N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid (HEPES), 3.5g of bovine serum albumin, 3.7g of sodium lactate, and 1.0g of glucose and dissolve them in 800ml of distilled water. Stir to dissolve;

[0060] 2) Use 0.5mol / L NaOH solution to adjust the pH to 7.5, distill the volume to 1000ml with distilled water, which is the capacitation buffer, store at 4°C, and each kit only needs 60ml.

[0061] 2. Preparation of fixative

[0062] Weigh 100ml of methanol as the fixative of this kit.

[0063] 3. Preparation of Fluorescent Conjugates

[0064] Use 1.0ml of tyrosine phosphorylated antibody-FITC with a concentration of 0.5mg / ml and 99ml of phosphate buffer solution with a concentration of 0.01mol / L and pH8.0 to prepare a tyrosine phosphorylated antibody-FITC concentration of 100ml of 5mg / L phosphate buffer is the fluorescent conjugate, and only 2ml is needed for each kit. Th...

Embodiment 2

[0100] 1. Kit preparation

[0101] 1. Preparation of capacitation buffer

[0102] 1) Weigh 0.4766g of N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid (HEPES), 0.35g of bovine serum albumin, 0.37g of sodium lactate, and 0.1g of glucose and dissolve them in 800ml of distilled water. Stir to dissolve;

[0103] 2) Use 0.01mol / L NaOH to adjust the pH to 7.0, distill the volume to 1000ml with distilled water, which is the capacitation buffer, store at 4°C, and only 60ml is needed for each kit.

[0104] 2. Preparation of fixative

[0105] Weigh 90ml of ethanol and dilute to 100ml with distilled water as the fixative solution for each kit.

[0106] 3. Preparation of Fluorescent Conjugates

[0107]Use 1.0ml of tyrosine phosphorylated antibody-FITC with a concentration of 0.5mg / ml and 499ml of phosphate buffer solution with a concentration of 0.01mol / L and pH8.0 to prepare a tyrosine phosphorylated antibody-FITC concentration of 500ml of 1mg / L phosphate buffer is the fluoresce...

Embodiment 3

[0140] 1. Kit preparation

[0141] 1. Preparation of capacitation buffer

[0142] 1) Weigh 23.83g of N-(2-hydroxyethyl)piperazine-N-2-ethanesulfonic acid (HEPES), 20.0g of bovine serum albumin, 20.0g of sodium lactate, and 10.0g of glucose and dissolve them in 800ml of distilled water. Stir to dissolve;

[0143] 2) Use 0.5mol / L NaOH to adjust the pH to 9.0, distill the volume to 1000ml with distilled water, which is the capacitation buffer, store at 4°C, and only 60ml is needed for each kit.

[0144] 2. Preparation of fixative

[0145] Weigh 100ml of acetic acid as the fixative for each kit.

[0146] 3. Preparation of Fluorescent Conjugates

[0147] Use 1.0ml of 0.5mg / ml tyrosine phosphorylated antibody-FITC and 49ml of 0.01mol / L, pH8.0 phosphate buffer to prepare 10mg of tyrosine phosphorylated antibody-FITC 50ml of phosphate buffer per L is the fluorescent conjugate, and each kit only needs 2ml. The tyrosine phosphorylated antibody-FITC is Mouse Anti-Phosphotyrosine FI...

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Abstract

The invention provides a detection kit of phosphorylation of sperm tyrosine. The detection kit comprises the following components: a capacitation buffer liquid, a fluorescent combined object and a fluorescent solid sealing object. The invention also provides a method for detecting the phosphorylation of the sperm tyrosine by using the kit. Compared with the prior art, according to the invention, each link of methodology is optimally designed, which includes the best buffer liquid matching of capacitation and an upstream method, a stable reaction place offering by special tissue carrier sheets, sperm double-labeling technique formulation combined with specificity, and influences to the detection result by non-sperm substances of cast-off cells in a seminal fluid are eliminated, thereby guaranteeing the methodology reliability, detection result specificity and good repeatability. Because an optimized detection scheme is adopted, the operation steps and the operation time are reduced, and the detection efficiency is improved greatly; because the optimized detection scheme is adopted, the reagent structure is simplified, and the detection cost is lowered to a greater degree; and because the operation is simple, ready-to-use available reagents are adopted, and the performance is stable.

Description

technical field [0001] The invention belongs to the category of in vitro detection reagents, in particular to a detection kit for sperm tyrosine phosphorylation and a detection method thereof. Background technique [0002] Sperm and oocyte binding initiates the acrosome reaction, releasing free soluble acrosomal components and exposing bound soluble acrosome components, driven by hyperactivated flagellar motility to enable sperm to penetrate the egg with matrix. To test this process, human oocytes derived from autopsy, surgical ovariectomy, or in vitro fertilization are used. These assays can use oocytes stored in saline, but the assays are limited by the scarcity of human oocytes. [0003] The existing technology uses hamster eggs instead of human oocytes as a screening method. This method has the following disadvantages: ① hamster breeding is cumbersome; ② hamster egg retrieval and follow-up operations are complicated; ③ detection scheme reagents are not perfect; ④ detec...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 张翔程浩虞少峰邓彩容程芳熊桃
Owner BRED LIFE SCI TECH
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