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Human KIF22(Kinesin-like protein KIF22) ELISA Kit (AEFI01096)

SKU:
AEFI01096
Product Type:
ELISA Kit
Size:
96T
Sensitivity:
18.75pg/ml
Range:
31.25-2000pg/ml
ELISA Type:
Sandwich ELISA
Reactivity:
Human
$719
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Description

Product Name: Human KIF22(Kinesin-like protein KIF22) ELISA Kit
Product Code: AEFI01096
Size: 96T
Alias: KIF22 ELISA Kit, Kinesin-like protein KIF22 ELISA Kit
Detection method: Sandwich ELISA, Double Antibody
Reactivity: Human
Sample Type: Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples
Detection Wavelength: OD450
Reaction Duration: 4 hours
Specificity: Specifically binds with KIF22 , no obvious cross reaction with other analogues.
Sensitivity: 18.75pg/ml
Range: 31.25-2000pg/ml
Storage: 2-8°C please do not freeze. See kit label for expiry date
Note: For Research Use Only
Recovery:
Matrix Recovery range(%) Average(%)
Serum(n=5) 86-102 95
EDTA Plasma(n=5) 88-100 91
Heparin Plasma(n=5) 87-102 94
Linearity:
Sample 1:2 1:4 1:8
Serum(n=5) 86-103% 87-97% 86-100%
EDTA plasma(n=5) 86-105% 90-100% 86-98%
Heparin Plasma(n=5) 93-105% 92-100% 87-100%
CV(%): Intra-Assay: CV<8%
Inter-Assay: CV<10%

This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti KIF22 antibody was pre-coated onto the 96-well plate. The biotin conjugated anti KIF22 antibody was used as the detection antibody. The standards and pilot samples were added to the wells subsequently. After incubation, unbound conjugates were removed by wash buffer. Then, biotinylated detection antibody was added to bind with KIF22 conjugated on coated antibody. After washing off unbound conjugates, HRP-Streptavidin was added. After a third washing, TMB substrates were added to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. The concentration of KIF22 in the sample was calculated by drawing a standard curve. The concentration of the target substance is proportional to the OD450 value.

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.

Sample Type Protocol
Serum Place whole blood sample at room temperature for 2 hours or at 2-8°C overnight. Centrifuge for 20min at 1000xg and collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay.
Plasma EDTA-Na2/K2 is recommended as the anticoagulant. Centrifuge samples for 15 minutes at 1000×g 2-8°C within 30 minutes after collection. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay. For other anticoagulant types and uses, please refer to the sample preparation guideline.
Tissue Sample

Generally tissue samples are required to be made into homogenization. Protocol is as below:

3.1. Place the target tissue on the ice. Remove residual blood by washing tissue with pre-cooling PBS buffer (0.01M, pH=7.4). Then weigh for usage.

3.2. Use lysate to grind tissue homogenates on the ice. The adding volume of lysate depends on the weight of the tissue. Usually, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS (e.g. 1mM PMSF).

3.3. Do further process using ultrasonic disruption or freeze-thaw cycles (Ice bath for cooling is required during ultrasonic disruption; Freeze-thaw cycles can be repeated twice.) to get the homogenates.

3.4. Homogenates are then centrifuged for 5 minutes at 5000×g. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -20°C or -80°C for future’s assay.

3.5. Determine total protein concentration by BCA kit for further data analysis. Usually, total protein concentration for Elisa assay should be within 1-3mg/ml. Some tissue samples such as liver, kidney, pancreas which containing a higher endogenous peroxidase concentration may react with TMB substrate causing false positivity. In that case, try to use 1% H2O2 for 15min inactivation and perform the assay again.

Notes: PBS buffer or the mild RIPA lysis can be used as lysates. While using RIPA lysis, make the PH=7.3. Avoid using any reagents containing NP-40 lysis buffer, Triton X-100 surfactant, or DTT due to their severe inhibition for kits’ working. We recommend using 50mM Tris+0.9%NaCL+0.1%SDS, PH7.3. You can prepare by yourself or contact us for purchasing.

Cell culture supernatant Collect the supernatant: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes, then collect clarified cell culture supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.
Cell lysates

5.1. Suspension Cell Lysate: Centrifuge at 2500 rpm at 2-8℃ for 5 minutes and collect cells. Then add precooling PBS into collected cell and mix gently. Recollect cell by repeating centrifugation. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Lyse the cell on ice for 30min-1h or disrupt the cell by ultrasonic disruption.

5.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS to wash three times. Add 0.5-1ml cell lysate and appropriate protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape the adherent cell with cell scraper. Lyse the cell suspension added in the centrifuge tube on ice for 30min-1h or disrupt the cell by ultrasonic disruption.

5.3. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Mucilaginous product is DNA which can be disrupted by ultrasonic cell disruptor on ice. (3~5mm probe, 150-300W, 3~5 s/time, 30s intervals for 1~2s working).

5.4. At the end of lysate or ultrasonic disruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.

Other Biological Sample

Centrifuge samples for 15 minutes at 1000×g at 2-8℃. Collect the supernatant to detect immediately. Or you can aliquot the supernatant and store it at -80°C for future’s assay.

Recommended reagents for sample preparation: Cat No: E051 100mM PMSF protease inhibitor, Cat No: E050 FineTest Lysis Buffer (for ELISA).

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