고양이 번호: BC3830

크기: 50T/48S

저장： The reagents are transported at room temperature, stored as required after arrival, and stable within 0.5 연령.

Product Composition

시약Ⅰ: 20 밀리리터×1. -20℃에서 보관；

시약 Ⅱ: 60 밀리리터×1. -20℃에서 보관.

시약 Ⅲ: 0.55 밀리리터×1. -20℃에서 보관, 빛을 피하다. 5mM pNA standard: 1 밀리리터×1. -20℃에서 보관, 빛을 피하다.

Preparation of Standard Diluent: 가져가다 9 mL of Reagent Ⅰ and add 1 mL of Reagent Ⅱ, 잘 섞다, and wait for use. (it can also be prepared according to the ratio of Reagent Ⅰ: Reagent Ⅱ = 9:1).

제품 설명:

Caspase is a family of proteases involved in the process of apoptosis, including more than 10 members. Caspase-3 is the most important terminal protease in the process of apoptosis, and it is also the most studied caspase; it activates pro-caspase-2,6,7,9, specifically hydrolyzes a variety of key apoptotic proteins, such as PARP, and mediates chromatin condensation, apoptotic body formation, and nuclear DNA fragmentation.

The caspase-3 colorimetric assay is based on the hydrolysis of the peptide substrate DEVD-pNA (Asp- Glu-Val-Asp-p-nitroanilide) by caspase-3, resulting in the release of the p-nitroaniline (pNA) moiety. p- Nitroaniline has a high absorbance at 405 nm. The activity of Caspase can be calculated by detecting pNA. This kit is suitable for mammalian tissue and cells.

필요하지만 제공되지 않는 시약 및 장비:

Spectrophotometer/Microplate reader, 100μL cuvette/ 96-well plate, 원심분리기, water bath/incubator, 조절 가능한 피펫, 모르타르/균질화기, ice, 그리고 증류수.

절차:

ㅏ. 샘플 준비:

1. Cells: collect the cells into the centrifuge tube, centrifuge and discard the supernatant; add 100μL Reagent Ⅱ to the number of cells (~에 대한 106 세포), shake and resuspend the precipitate, then stand on ice for15 min, centrifuge 15000g at 4℃ for 10 15 분, take the supernatant and place it on ice for (it can be increased to 150-200 μL Reagent Ⅱ if the cracking is not enough)
2. 조직: according to the ratio of tissue mass (g): Reagent Ⅱ volume (밀리리터) ~의 1:5-10 (무게를 측정하는 것이 좋습니다 0.1 g 티슈 추가 1 mL of Reagent Ⅱ), grind it in an ice bath or cut it thoroughly, place it on ice for 15 분, centrifuge it at 4℃ for 10-15 분, 상등액을 채취하여 얼음 위에 올려 놓고 테스트해 보세요..

비. 결정 절차:

1. Preheat the spectrophotometer/microplate reader for 30 분, 파장을 조정하여 405 nm, and adjust the distilled water to zero.
2. 사용하기 전에, 5 mmol/L PNA standard solution is diluted to 200, 100, 50, 25, 5, 그리고 0 μmol/L standard solution with standard solution diluent.
3. Sample determination (add the following reagents in sequence in 96 well plate / EPtube)

씨. Activity 계산:

1. Establishment of the standard curve

The standard equation is made according to the concentration of the standard tube (엑스, μmol/L) and ΔAS (y, minus the tube with 0 집중). The determination of ΔAT is substituted into the standard equation to obtain x (μmol/L).

2. According to the increased percentage of enzyme activity

Increased percentage of caspase-3 activity = ((experimental treatment group AT)- AB) / ((experimental control group AT)- AB) × 100%

The method is simple and reliable and can be used to determine the enzyme activity roughly.

3. Calculated by enzyme activity

One unit is the amount of enzyme that will cleave 1.0 nmol of the colorimetric pNA-substrate per hour at 37℃ under saturated substrate concentrations. we can calculate the caspase activity in the sample.

Caspase-3 activity (U/mg 프로트) = x × VR ÷ (VS × Cpr ) ÷ T × 103 = 2x ÷ Cpr ÷ t

VR: total volume of the reaction system, 0.1 mL = 10-4 엘; VS: volume of added sample, 0.05 밀리리터; 티: 반응 시간, 1 시간; 심폐소생술: concentration of sample protein, mg/mL; 103: unit conversion coefficient, 1 μmol = 103 nmol.

메모:

1. Since Reagent I contains a reducing agent (DTT), it is recommended to dilute the sample 2 times with distilled water and then use the Bradford method to determine the protein concentration to reduce the interference of DTT on the protein concentration determination. It is not recommended to use the BCA method to determine protein
2. The most common reason for the low Caspase activity value is that the cells have not undergone apoptosis, the amount of cells is too small or the observation time is When inducing apoptosis, it is not that the larger the dose, the longer the time, the higher the Caspase activity. It is recommended to set different doses and time points such as 0, 2, 4, 8, 16, 그리고 24 hours to detect the best observation point.
3. When the value of the measured sample is higher than the upper limit of the standard curve, the sample can be diluted with Reagent Ⅱ and then re-measured.
4. Tightly cover the 96-well plate and seal it with parafilm. 배양 장소 37 ºC, the OD405 value when the color turns yellow is about 0.2, which can be measured at this time. The insignificant color change can prolong the reaction or overnight, but when the enzyme activity is strong, too long incubation time will cause the reaction to lose the linear

Recent Product Citations：

• Wang B , Wang K , Jin T , 외. NCK1-AS1 enhances glioma cell proliferation, radioresistance, and chemoresistance via the miR-22-3p/IGF1R ceRNA pathway[제이]. Biomedicine & Pharmacotherapy, 2020, 129:110395.
• Wang Z , Xu J H , Mou J J , 외. Novel ultrastructural findings on cardiac mitochondria of huddling Brandt’svoles in a mild cold environment[제이]. Comparative Biochemistry and Physiology – Part A Molecular & Integrative Physiology, 2020, 249:110766.

참고자료:

• Cohen GM. Caspases: the executioners of apoptosis. Biochem J, 1997, 326:1-16.
• JanickeR U, Sprengart M L, Wati M R, et Emerging role of caspase-3 in apoptosis[제이]. Cell Death and Differentiation, 1999, 6:99-104.

관련 상품:

BC3810 Caspase-1 activity assay Kit BC3820 Caspase-2 activity assay Kit BC3840 Caspase-4 activity assay Kit BC3850 Caspase-5 activity assay Kit BC3860 Caspase-6 activity assay Kit BC3880 Caspase-8 activity assay Kit BC3890 Caspase-9 activity assay Kit