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
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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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