Rat Apoptosis Inducing Factor (AIF) ELISA Kit

Rat Apoptosis Inducing Factor (AIF) ELISA Detection Kit

Detection Principle :

This kit uses a double antibody sandwich enzyme-linked immunosorbent assay（ ELISA）。 Pre packaged Rat apoptosis inducing factor (AIF)Add the solid-phase antibody to the microporous enzyme-linked immunosorbent assay (ELISA) plate Rat apoptosis inducing factor (AIF) Calibrate and test samples, then add another strain HRP labeled antibody Rat apoptosis inducing factor (AIF) (Enzyme labeled antibody), after incubation and thorough washing, unbound components are removed to form solid-phase antibodies on the solid surface of the microplate -A sandwich complex of antigen enzyme-linked antibody. Add substrates A and B, and under the catalysis of HRP, the substrates produce a blue product. Under the action of termination solution (2M sulfuric acid), it is finally converted to yellow. Measure the absorbance (OD value) at a wavelength of 450nm on an enzyme-linked immunosorbent assay (ELISA) reader, and compare the absorbance (OD value) with the sample to be tested Rat apoptosis inducing factor (AIF)The concentration is positively correlated. Fitting the calibration curve can calculate the concentration in the sample Rat (AIF)The concentration.

Required equipment and reagents

1 Microplate Reader (450nm wavelength filter)

237 ° C constant temperature incubator (CO2 incubator for cells is not recommended)

3 Automatic washing machine or multi-channel pipette /5ml dropper (for manual board washing)

4 sterile EP tube and disposable suction head

5 Precise single track (0.5-10 μ L, 5-50 μ L, 20-200 μ L, 200-1000 μ L) and multi-channel pipettes (pipettes need to be calibrated before use).

6Absorbent paper and sample tank

7Deionized water or distilled water

Kit composition:

Sample collection :

1、The test tube for collecting blood should be a disposable, pyrogen free, endotoxin free tube.

2、 Recommended use of plasma anticoagulants EDTA 。 Avoid using hemolytic and hyperlipidemic specimens.

3、The specimen should be clear and transparent, and suspended solids should be removed by centrifugation.

4、 If the specimen is not tested in a timely manner after collection, please pack it according to the one-time usage amount and freeze it in -Inside the refrigerator at 20 ℃ and -70 ℃, avoid repeated freezing and thawing, and test within 3-6 months.

5、 If the concentration of the test substance in your sample is higher than the highest value of the standard, please adjust according to the actual situation, Dilute appropriately by multiple (It is recommended to conduct preliminary experiments to determine the dilution factor).

Storage conditions:

Sample collection and preservation：

1 Serum and whole blood samples are stored at room temperature 2 hours or overnight at 2-8 ° C. Centrifuge at 1000 × g for 20 minutes and collect the supernatant. Can be detected immediately, or packaged and frozen at -20 ° C or -80 ° C according to a single usage amount.

2 Recommended use of plasma anticoagulants EDTA-Na2/K2， Centrifuge at 2-8 ° C and 1000 × g for 15 minutes after sample collection, and collect the supernatant. Can be tested immediately, or packaged and frozen at -20 ° C or -80 ° C according to a single use amount. For the use and selection of other anticoagulants, please refer to the sample preparation guide.

3The tissue sample is generally made into tissue homogenate, and the processing method is as follows:
3.1 Place the target organization on ice and use pre cooled Wash with PBS buffer (0.01M, pH=7.4) to remove residual blood, weigh and set aside.

3.2 Grind the tissue homogenate with lysis buffer on ice. The volume of added lysis buffer depends on the weight of the tissue, and generally, each 1g of tissue fragments were treated with 9ml of lysis buffer. Additionally, it is recommended to add protease inhibitors such as 1mM PMSF to the lysis buffer.

3.3 Can be further processed by ultrasonic crushing or repeated freeze-thaw cycles During the ultrasonic crushing process, an ice bath is required to cool down; the repeated freeze-thaw method can be repeated twice.

3.4 Mix the prepared homogenate in Centrifuge at 5000 × g for 5 minutes and collect the supernatant for detection. Alternatively, it can be packaged and frozen at -20 ° C or -80 ° C according to a single usage amount.

3.5 According to experimental requirements, the total protein concentration of the tissue homogenate sample can be measured first To facilitate data analysis, the BCA method is recommended. Generally, adjust the total protein concentration to 1-3mg/ml for ELISA detection. Some tissue samples, such as liver, kidney, and pancreas, may react with chromogenic substrates at high concentrations due to the presence of endogenous peroxidase, resulting in false positives. You can try using 1% H2O2 to inactivate for 15 minutes before testing.

Note: Common use of cracking solution PBS buffer or use medium strength RIPA lysis buffer. When in use, the pH value should be adjusted to pH 7.3 to avoid using components containing NP-40, Triton X-100, and DTT, which can severely inhibit the operation of the reagent kit. We recommend using 50mM Tris+0.9% NaCl+0.1% SDS, pH 7.3. You can prepare it yourself or contact us for purchase.

4 Collect the supernatant from cell culture, Centrifuge at 2-8 ° C and 2500rpm for 5 minutes, and collect clear cell culture supernatant. Immediately used for testing, or packaged in a single use amount and stored at -80 ° C for future use.

5cell lysate

5.1 Collection and lysis of suspended cells: Centrifuge at 2-8 ° C and 2500rpm for 5 minutes to collect cells. Add pre cooled PBS, gently mix and wash, centrifuge at 2-8 ° C and 2500rpm for 5 minutes, and collect cells. Add 0.5-1ml of cell lysis buffer and an appropriate amount of protease inhibitor (such as PMSF, working concentration 1mmol/L), place on ice, lyse for 30-1h, or combine with ultrasonic disruption.

5.2 Collection and lysis of adherent cells: Remove the supernatant and add pre cooled Wash with PBS three times. Add 0.5-1ml of cell lysis buffer and an appropriate amount of protease inhibitor (such as PMSF, working concentration 1mmol/L), and gently scrape off the adherent cells with a cell scraper. Transfer the cell suspension into a centrifuge tube, place it on ice, lyse for 30 minutes to 1 hour, or combine with ultrasonic fragmentation.

5.3 During the process of cell lysis, the centrifuge tube can be blown with a gun or shaken intermittently to fully lyse the protein The appearance of sticky DNA can be broken by ultrasound. Alternatively, use an ultrasonic probe with a diameter of 3-5mm and a power of 150-300W to treat the sample on ice. Operate for 1-2 seconds, stop for 30 seconds, and repeat 3-5 cycles. )

5.4 Cracking or ultrasonic fragmentation is completed, Centrifuge at 2-8 ° C and 10000rpm for 10 minutes. Transfer the supernatant into an EP tube and immediately use it for detection, or divide it into single use portions and store at -80 ° C for later use.

Rat Apoptosis Inducing Factor (AIF) ELISA Detection Kit

Note:The precautions are the same as those for organizing samples.

Note: The above is for reference only! I hope the above information can help you make a suitable choice!