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Rat Cytochrome b (MT-CYB) ELISA Kit BHE11404002

BHE11404002
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+1 866.986.9598
Email
[email protected]
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Uniprot P34863
Description The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition.
The loss rate was determined by accelerated thermal degradation test. Keep the kit at 37°C for 4 and 7 days, and compare O.D.values of the kit kept at 37°C with that of at recommended temperature. (referring from China Biological Products Standard, which was calculated by the Arrhenius equation. For ELISA kit, 4 days storage at 37°C can be considered as 6 months at 2 - 8°C, which means 7 days at 37°C equaling 12 months at 2 - 8°C).
Storage Store at 2-8°C. Please refer to Instruction Manual.
Expiry Date Half Year
Applications ELISA
Protocols Description Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
Other Protocol Bring all reagents and samples to room temperature before use. It is recommended that all samples, controls, and standards be assayed in duplicate.
1.Prepare all reagents, working standards, and samples as directed in the previous sections.
2.Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 2 - 8°C.
3.Add 100 µL of standard and sample per well. Cover with the adhesive films provided. Incubate for 2 hours at 37°C. A plate layout is provided to record standards and samples assayed.
4.Aspirate each well and wash, repeating the process for a total of three washes. Wash by filling each well with Wash Buffer (250 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5.Add 100 µL of Biotin-Conjugate (1 x) to each well. Cover with the adhesive films. Incubate for 1 hour at 37°C. (Biotin-Conjugate (1 x) may appear cloudy. Warm up to room temperature and mix gently until solution appears uniform.)
6.Aspirate each well and wash, repeating the process for a total of three washes. Wash by filling each well with Wash Buffer (250 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
7.Add 100 µL of Streptavidin-HRP (1 x) to each well. Cover the microtiter plate with the adhesive films. Incubate for 1 hour at 37°C.
8.Aspirate each well and wash, repeating the process for a total of five washes. Wash by filling each well with Wash Buffer (250 µL) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
9.Add 100 µL of Substrate Solution to each well. Incubate for 15-20 minutes at 37°C. Keeping the plate away from drafts and other temperature fluctuations in the dark. Avoid placing the plate in direct light.
10.Add 50 μL of Stop Solution to each well. When the first four wells containing the highest concentration of standards develop obvious blue color. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
11.Determine the optical density of each well within 5 minutes, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. Subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.

Serum: Use a serum separator tube (SST) and allow samples to clot for two hours at room temperature or overnight at 2 - 8°C before centrifugation for 15 minutes at 1000 × g. Remove serum and assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Plasma: Collect plasma using EDTA, or heparin as an anticoagulant. Centrifuge for 15 minutes at 1000 × g at 2 - 8°C within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20°C. Avoid repeated freeze-thaw cycles. Other biological fluids: Centrifuge samples for 20 minutes at 1000 × g. Remove particulates and assay immediately or store samples in aliquot at -20°C or -80°C. Avoid repeated freeze/thaw cycles.
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