Kit Uji Aktivitas Solarbio Ca++Mg++-ATPase

Ca++Mg++-ATPase Activity Assay Kit

Catatan: Ambil dua atau tiga sampel berbeda untuk prediksi sebelum pengujian.

Peralatan Operasi: Spektrofotometer

Kucing No: BC0960

Ukuran: 50T/24S

Komponen:

Reagen I: Cairan 30 mL×1. Penyimpanan pada suhu 4℃.

Reagen II: Cairan 4 mL×1. Penyimpanan pada suhu 4℃.

Reagen III: Bedak×2. Storage at -20℃. Dissolve thoroughly with 1 mL air suling sebelum digunakan. The rest reagent can be kept at -20℃ for one week.

Reagen IV: Cairan 2 mL×1. Penyimpanan pada suhu 4℃.

Reagen V: Bedak×1. Penyimpanan pada suhu 4℃. Dissolve thoroughly with 3 mL air suling sebelum digunakan. Reagent VI: Bedak×1. Penyimpanan pada suhu 4℃. Dissolve thoroughly with 15 mL air suling sebelum digunakan, can be kept at 4℃ for one week.

Reagent VII: Bedak×1. Penyimpanan pada suhu 4℃. Dissolve thoroughly with 15 mL air suling sebelum digunakan, can be kept at 4℃ for one week.

Reagent VIII: Cairan 15 mL×1. Storage at RT.

Solusi standar: Cairan 1 mL×1. 10 μmol/mL standard phosphorus liquid, storage at 4℃.

0.5 μmol/mL standard phosphorus working solution: Dilute the 10 μmol/mL standard 20 times with distilled water to 0.5 μmol/mL standard. Misalnya: menambahkan 1.9 mL air suling ke 0.1 mL of standard, aduk rata.

Phosphorus fixing reagent:

Prepare reagents for determining phosphorus content: make solution as the volume ratio of H2O: Reagent VI: Reagent VII: Reagent VIII =2:1:1:1, which should be light yellow. It shows lose efficacy if color is changed, phosphorus pollution if color is change to blue. Prepare the reagent when it will be used.

Catatan: It is better to use new beakers, glass rods and glass pipettes or disposable plastic ware when making reagent to avoid phosphorus pollution.

Deskripsi Produk:

Ca++Mg++-ATPase is widely distributed in plants, animals, microorganisms and cells, which catalyzes the hydrolysis of ATP to form ADP and inorganic phosphorus.

Ca++Mg++-ATPase decomposes ATP to produce ADP and inorganic phosphorus. The activity of ATPase can be detected by measuring the amount of inorganic phosphorus.

Reagen dan Peralatan Diperlukan tetapi Tidak Disediakan:

Spektrofotometer, mesin sentrifugal meja, pipet yang dapat disesuaikan, mandi air, 1 mL kuvet kaca, mortir/homogenizer, es dan air suling.

Prosedur:

SAYA. Persiapan sampel:

1. Bakteri atau sel:

Collecting bacteria or cells into a centrifuge tube, centrifugation, and discard supernatant. Suggest add 1mL of Reagent I to 5 million of bacteria or cells. Use ultrasonic to splitting bacteria and cells (ditempatkan di atas es, ultrasonic power 20%, working time 3 detik, selang 10 detik, ulangi untuk 30 waktu). Sentrifugasi di 8000 ×g untuk 10 minutes at 4℃ and take the supernatant on ice before testing.

2. Jaringan:

Menambahkan 1 mL of Reagent I into 0.1 g tisu, fully grinding on ice. Sentrifugasi di 8000 ×g untuk 10 minutes at 4℃ and take the supernatant on ice before testing.

3. Serum: Directly

II. Tekad:

1. Preheat spectrophotometer for 30 menit, adjust the wavelength to 660 nm, set the counter to zero with distilled water.
2. Add the following reagents to EP tube:

3. Determination of phosphorus content, add the following reagents to 1.5 mL EP tube:

Aduk rata, then place the mix solution in a 40℃water bath for 10 menit. Cooling to room temperature and detect the absorbance at 660 nm. The blank tube and standard tube just need one or two tubes.

AKU AKU AKU. Perhitungan:

1. Serum:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milliliter of serum.

Ca++Mg++-ATPase (U/mL)=Cs×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]×Vrv÷s÷T

=7.5×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]

2. Jaringan, bacteria, or cells

• Konsentrasi protein:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every milligram of tissue protein.

Ca++Mg++-ATPase (U/mg keuntungan)=Cs×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]×Vrv ™(Vs×Cpr)÷T

=7.5×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]−Cpr

• Berat sampel:

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour, every milligram of tissue.

Ca++Mg++-ATPase (berat U/g)=Cs×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]×Vrv ™(Vs÷V1×W)÷T

=7.5×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]−W

• bacteria or cells

Unit definition: One unit of enzyme activity is defined as the amount of enzyme catalyzes the decomposed of ATP to produce 1 μmol of inorganic phosphorus per hour every 10000 sel atau bakteri.

Ca++Mg++-ATPase (U/104cell )=Cs×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]×Vrv ™(Vs÷V1×500)÷T

=0.015×[ΔA(T)-ΔA(C)]−[ΔA(S)-ΔA(B)]

Cs: Concentrate of standard tube, 0.5 μmol/mL;

Tali: Volume reaksi total, 0.5 ml; Vs: Volume sampel, 0.2 ml;

Cpr: Konsentrasi protein sampel (mg/mL); T: Waktu reaksi (menit), 1/6 jam;

W: Berat sampel (G);

Vl: Volume of reagent I, 1 ml;

500: The amount of bacteria or cell, 5 juta.

Catatan

1. This kit can detect 24 tubes of Ca++Mg++ -ATPase samples in 50 tubes for each sample need one tube as control.
2. This method has the characteristics of trace, sensitive and rapid. The test tubes used for determination are phosphate-free strictly. Avoiding phosphorus pollution is the key to the success of detection.

Contoh eksperimental:

1. Take of pancreas and add 1 mL of Reagent I for ice bath homogenization. After centrifugation at 4℃ for 10 menit, the supernatant is put on the ice and operated according to the determination steps. ΔAT = 0.916-0.389=0.527, ΔAS =0.398-0.004=0.394

Ca++Mg++- ATPase activity (massa U/g) = 7.5 × ΔAT ÷ΔAS ÷ W = 100.32 massa U/g.

2. Take 0.1g of willow and add 1 mL of Reagent Ⅰ for ice bath homogenization. After centrifugation at 4℃ for 10 menit, the supernatant is put on ice and operated according to the determination steps. The ΔAT=0.137-0.124=0.013, and the ΔAS=398-0.004=0.394

Ca + + Mg + + – ATPase activity (massa U/g) = 7.5×ΔAT ÷ ΔAS ÷ W = 2.47 massa U/g.

Referensi

[1] Datiles M J, Johnson E A, McCarty R E. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles[J]. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 2008, 1777(4): 362-368.

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