Experimental detection of apoptosis factor Caspase-389

Experimental detection of apoptosis factor Caspase-389

Caspase3

CaspaseIt is a protease family that plays an important role in the process of cell apoptosis. Caspase3, also known as CPP32, is a key enzyme in the process of cell apoptosis. It can cleave procaspase 2.6, 7, and 9, and can directly and specifically cleave many caspase substrates, including PARP (poly(ADP-ribose) polymerase), ICAD (Inhibitor of caspase-activated Deoxyri- bonuclease), gelsolin And fodrin and others. These protein cleavages mediated by caspase 3 are important components of the molecular mechanism of cell apoptosis. Caspase 3 also plays a key role in the process of nuclear apoptosis, including chromatin fixation

Shrinkage, fragmentation of DNA, etc. Meanwhile, caspase 3 also plays a crucial role in cell foaming.

The detection principle is based on the fact that casepase 3 can catalyze the substrate AC DEVD pNA to produce yellow pNA (- troaniline), allowing for the detection of caspase 3 activity by measuring absorbance. PNA exhibits strong absorption around 405nm. The standard curve prepared based on the yellow product pNA can be used as a quantitative standard for caspase 3 enzyme activity.

Caspase-8

Caspase 8 usually exists in the form of a zymogen and is activated during the signal transduction process of cell apoptosis. Caspase 8 is considered an upstream caspase in the signal transduction process of cell apoptosis. In the process of cell apoptosis mediated by Fas receptor and TNFR-1, Caspase 8 is activated, forming a dimer composed of p18 and p10, which further activates downstream caspase 4 and caspase 6. Caspase 9 and caspase 10. This method is based on the fact that Caspase 8 can catalyze the substrate AC IETD pNA to produce yellow pNA (nitraniline), allowing for the detection of Caspase 8 activity by measuring absorbance. PNA exhibits strong absorption around 405nm. The 405nm OD value of the test sample can be calculated based on the standard curve prepared by pNA to determine the Caspase 8 enzyme activity of the sample.

Caspase-9

Caspase 9, also known as ICE-LAP6 or Mch6, is a cellapoptosisThe upstream caspase in the signal transduction process. After mitochondria release cytochrome c, caspase 9 can form a complex with cytochrome c and Apaf1 and be activated simultaneously. Activated caspase 9 can activate the key enzyme caspase 3 in cell apoptosis, thereby promoting subsequent apoptotic signaling. The activation of Caspase 9 can be regulated through phosphorylation.

This method is based on Caspase 9, which can catalyze the substrate Ac LEHD pNA to produce yellow pNA (p-nitroaniline), which can be detected by measuring absorbance

The activity of Caspase 9. PNA exhibits strong absorption around 405nm. The 405nm OD value of the test sample can be calculated based on the standard curve prepared by pNA to determine the Caspase 9 enzyme activity of the sample.

Caspase1

Caspase2

Caspase4

Caspase6

Apoptosis factor Caspase3/8/9 Sample preparation for testing:

1) Cell sample: Take 1x107 cells (approximately 1 T25 culture bottle), centrifuge 600g at 4 ° C for 5 minutes to collect cells, carefully aspirate the supernatant, and wash once with PBS. After absorbing the supernatant, 100u of lysis buffer was added to each tube of cell samples to resuspend the precipitate, which was then lysed in an ice bath for 15 minutes. Centrifuge at 4 ° C 16000g for 10-15 minutes, transfer the supernatant to a pre cooled centrifuge tube in an ice bath. Immediately measure the enzyme activity of caspase 3 or store the sample at -70 ° C. At the same time, a small amount of sample can be taken to determine protein concentration using the Bradford method, aiming to achieve a protein concentration of 1-3mg/ml

2) Organizational sample: Add 100ul of lysis buffer to approximately 10mg of tissue and homogenize on an ice bath using a glass homogenizer. Then transfer the homogenate to a 1.5ml centrifuge tube and lyse in an ice bath for another 5 minutes. Centrifuge at 4 ° C 16000g for 10-15 minutes, transfer the supernatant to a pre cooled centrifuge tube in an ice bath. Immediately measure the enzyme activity of caspase 3 or store at -70 ° C

Sample. At the same time, a small amount of sample can be taken to determine protein concentration using the Bradford method, aiming to achieve a protein concentration of 1-3mg/ml

Caspase-3 activity detection standard curve

Experimental period: Completed within 7-10 working days, with repeat testing discounts.

Customer precautions

1. Antibodies: Consult in advanceShanghai YanjinBiology, identify the species of antibody and whether the antibody is available for use. If there is no agreement between the two parties, the customer can purchase and provide it themselves or our company can act as a purchasing agent;

2. Specimen collection and preservation:

① Organizational samples: Fresh tissues are stored in liquid nitrogen or at 70 degrees Celsius, with a minimum of 100mg of tissue (approximately the size of a mung bean);

② Cultivate cells: Take no less than 000000 cells through cell counting and transfer them to an EP tube. Add 0.5ml of physiological saline or protein protectant, mix well, and store in a refrigerator (-70 ° C to avoid repeated freezing and thawing);

③ Blood cell specimen: Store the blood sample in an anticoagulant tube or separate the cells in lymphocyte isolation solution and add 0.5ml of physiological saline or protein protectant for preservation (can be stored for a long time at -70 ° C to avoid repeated freezing and thawing)

④ Serum or cell culture medium specimens: After centrifugation to remove impurities, take the supernatant and transfer it into an EP tube for storage (above 4 ° C for 15-20 days, -70 ° C for long-term storage to avoid repeated freezing and thawing)

3. Sample transportation: You can choose any of the following methods to send the specimen

① Organizational samples and cell samples are transported by dry ice or added with protein protectants and then transported in ice packs;

② Cell samples can also be sent directly to the culture bottle by express delivery (sealed to ensure no contamination, no leakage of culture medium, cells should not grow too full, about 50%, filled with culture medium);

③ Serum or supernatant can be directly sent in ice packs (sealed to ensure that the liquid does not leak, depending on the distance of the journey, it can take 2-3 days to arrive).

Experimental outsourcing service: