Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit
ELISA Genie Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit
The ELISA Genie Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit can assay for Human Cell division cycle protein 23 homolog in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.
How do our Human Cell division cycle protein 23 homolog (CDC23) ELISA Kits work?
The ELISA Genie Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit is based on the competitive binding enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Human Cell division cycle protein 23 homolog. During the reaction, in the sample or standard competes with a fixed amount of biotin-labeled for sites on a pre-coated Monoclonal antibody specific to Human Cell division cycle protein 23 homolog. Excess conjugate and unbound sample or standard are washed from the plate. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of in the samples is then determined by comparing the O.D. of the samples to the standard curve.Gene name
Cell division cycle protein 23 homolog, Anaphase-promoting complex subunit 8, APC8, Cyclosome subunit 8, CDC23, ANAPC8
Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit Protocol
The below protocol is a sample protocol for Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human Cell division cycle protein 23 homolog (CDC23) present in their sample.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37?Cdirectly). All the reagents should be mixed thoroughly by gently swirling beforepipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and removeextra strips from microtiter plate. Removed strips should be resealed and stored at -20?C untilthe kits expiry date. Prepare all reagents, working standards and samples as directed in theprevious sections. Please predict the concentration before assaying. If values for these arenot within the range of the standard curve, users must determine the optimal sample dilutionsfor their experiments. We recommend running all samples in duplicate.
|1.||Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is addedwith Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid insidewall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided.Incubate for 120 minutes at 37?C.|
|2.||Remove the liquid from each well, don't wash. Add 100µL ofDetection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap theplate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent Aappears cloudy warm to room temperature until solution is uniform.|
|3.||Aspirate each well and wash, repeating the process three times. Wash by fillingeach well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at eachstep is essential. After the last wash, completely remove remaining Wash Buffer by aspiratingor decanting. Invert the plate and pat it against thick clean absorbent paper.|
|4.||Add 100µL of Detection Reagent B working solution to each well.Cover with the Plate sealer. Incubate for 60 minutes at 37°C.|
|5.||Repeat the wash process for five times as conducted in step 3.|
|6.||Add 90µL of Substrate Solution to each well. Cover with a new Plate sealerand incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can beshortened or extended according to the actual color change, but this should not exceed morethan 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.|
|7.||Add 50µL of Stop Solution to each well. If color change does not appear uniform,gently tap the plate to ensure thorough mixing.|
|8.||Determine the optical density (OD value) of each well at once, using amicro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheatthe instrument, and set the testing parameters.|
|9.||After experiment, store all reagents according to the specified storage temperaturerespectively until their expiry.|
Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||-20°C|
|Assay Diluent A||10mL||-20°C|
|Assay Diluent B||10ml||-20°C|
|Detection Reagent A||120ul||-20°C|
|Detection Reagent B||120ul||-20°C|
Other materials and equipment required:
The ELISA Genie Human Cell division cycle protein 23 homolog (CDC23) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.