| Usage | Instructions for Use:
1. Preparation:
A. After the lysate is dissolved, mixed well and placed on an ice bath for later use.
b. The test buffer is dissolved and mixed well and placed on an ice bath for later use.
2. Determination of pNA standard curve:
A. Preparation of standard diluent: Prepare an appropriate amount of standard diluent according to the ratio of 0.1 ml of lysate per 0.9 ml of detection buffer.
b. The pNA (10 mM) supplied in the kit was diluted to 0, 10, 20, 50, 100 and 200 μM with a standard diluent as a standard.
c. Take 100μl of each concentration and detect it with a microplate reader, or take an appropriate amount and detect it with a spectrophotometric detection cup with a capacity not exceeding 100μl to determine A405.
d. The actual absorbance due to pNA was calculated by subtracting the A405 of the blank control without pNA from the A405 of each standard, and a standard curve of pNA concentration versus A405 was created.
3. Collection of samples:
A. For suspended cells: Centrifuge the control sample that did not induce apoptosis and the sample that induced apoptosis, 600 g at 4 º C for 5 minutes to collect the cells, carefully aspirate the supernatant, and ensure that no cells are aspirated as much as possible, and wash once with PBS. After the supernatant was aspirated as before, the lysate was added at a ratio of 100 microliters of lysate per 2 million cells, the pellet was resuspended, and lysed in an ice bath for 15 minutes. Go down to step 3d.
b. For adherent cells: aspirate the cell culture medium for later use. The adherent cells were digested with trypsin and collected into a spare cell culture medium. Cells were collected by centrifugation at 600 g at 4 º C for 5 minutes, the supernatant was carefully aspirated, while ensuring that no cells were aspirated as much as possible, and PBS was washed once. After the supernatant was aspirated as before, the lysate was added at a ratio of 100 microliters of lysate per 2 million cells, the pellet was resuspended, and lysed in an ice bath for 15 minutes. Go down to step 3d.
c. For tissue samples: Lysate was added at a ratio of 100 microliters of lysate per 3-10 mg of tissue and homogenized with a glass homogenizer on an ice bath. The homogenate was then transferred to a 1.5 ml centrifuge tube and lysed in an ice bath for an additional 5 minutes.
d. Centrifuge at 4 º C 16,000-20,000 g for 10-15 minutes.
e. The supernatant was transferred to an ice bath precooled centrifuge tube.
f. Determine the enzyme activity of caspase 3/7 immediately or save the sample at-80 º C. At the same time, a small amount of sample can be taken to measure the protein concentration by Bradford method, and try to make the protein concentration reach 1-3mg/ml, which is equivalent to at least 10-30μg of protein per 10 microliters of sample to be tested. If there are fewer cells, the amount of cells can be appropriately increased.
4. Detection of Caspase 3/7 enzyme activity:
A. Remove an appropriate amount of Ac-DEVD-pNA (2 mM) and place it on an ice bath for later use.
b. The reaction system was set up as follows:
| Name | Blank control | Sample | | Assay buffer | 40 μl | 40 μl | | Sample to be measured | 0 μl | 50 μl | | Lysate | 50 μl | 0 μl | | Ac-DEVD-pNA (2 mM) | 10 μl | 10 μl | | Total Volume | 100 μl | 100 μl |
Note: When setting the reaction system, add the detection buffer first, then add the sample to be tested, and mix properly. Be careful to avoid bubbles during mixing. An additional 10 μl of Ac-DEVD-pNA (2 mM) was then added.
c. Add Ac-DEVD-pNA (2 mM) and mix well, taking care to avoid bubbles during mixing. Incubate at 37 º C for 60-120 minutes. A405 can be measured when the color change is obvious. If the color change is not obvious, the incubation time can be appropriately extended, or even overnight.
d. A405 of the sample minus A405 of the blank control is the absorbance generated by pNA catalyzed by caspase 3/7 in the sample. The amount of pNA catalytically produced in the sample can be calculated by comparing it with the standard curve obtained in step 1.
e. Refer to the definition of caspase 3/7 enzyme activity units from Chemicon: One unit is the amount of enzyme that will clear 1.0 nmol of the colorimetric substrate Ac-DEVD-pNA per hour at 37 º C under saturated substrate concentrations. That is, an enzyme activity unit is defined as the amount of caspase 3/7 enzyme that can cleave 1 nmol of Ac-DEVD-pNA to produce 1 nmol of pNA within one hour at 37 º C when the substrate is saturated. This allows to calculate how many enzyme activity units of caspase 3/7 are contained in the sample. Note: In the detection system of this kit, the initial concentration of the substrate is 0.2 mM, at which time the substrate is saturated. For many samples, the substrate is saturated within 2 hours of incubation at 37 º C; If the activity of caspase 3/7 enzyme in the sample is particularly high, the sample must be properly diluted with lysate before determination.
f. Use the Bradford method to detect the protein concentration in the sample to be tested (because the lysate contains a high concentration of DTT, it is not suitable to use the BCA method for protein concentration determination). In this way, the enzyme activity unit of caspase 3/7 contained per unit weight of protein in a sample can be calculated.
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| Description | caspase 3/7 Activity Assay Kit (caspase 3/7 Activity Assay Kit) is a kit for detecting caspase 3/7 enzyme activity or purified caspase 3/7 enzyme activity in cell or tissue lysates by spectrophotometry.
Caspase (Cysteine-requiring Aspartate Protease) is a family of proteases that play an important role in the process of apoptosis. caspase 3, also known as CPP32, Yama or apopain, sometimes written as caspase-3 or caspase 3, belongs to the CED-3 subfamily of the caspase family and is a key enzyme in the process of apoptosis. caspase 3 is the most studied caspase in mammalian cells. Both Caspase 3 and Caspase 7 can cleave the DEVD peptide sequence, so this kit can detect caspase3/7.
This kit is based on the fact that caspase 3/7 can catalyze the substrate Ac-DEVD-pNA to produce yellow pNA (p-nitroaniline), so that the activity of caspase 3/7 can be detected by measuring absorbance. pNA has strong absorption near 405nm.
This kit can detect 20 samples (20T) or 100 samples (100T) in addition to the standard curve when detected with a microplate reader or a spectrophotometric detection cup with a capacity not exceeding 100μl.
Product Components: | Name | 20T | 100T | | Lysate Lysis buffer | 8ml | 30ml | | Assay buffer | 8ml | 20ml | | Ac-DEVD-pNA (2 mM) | 200ul | 1ml | | pNA (10 mM) | 200ul | 1ml |
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