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|Sample:||Serum,plasma,urine,tissue,cell Culture Supernatant||Standard Curve Range:||0.05ng/ml - 15ng/ml|
sandwich elisa kit,
rat elisa kit
Customized Mouse Osteopontin OPN Sandwich ELISA Kit 96 Wells 48Wells
Standard Curve Range: 0.05ng/ml - 15ng/ml
Size: 96 wells
Storage: Store the reagents at 2-8°C. For over 6-month storage refer to the expiration date keep it at -20°C. Avoid repeated thaw cycles. If individual reagents are opened it is recommended that the kit be used within 1 month.
*This product is for research use only, not for use in diagnosis procedures. It’s highly recommend to read this instruction entirely before use.
This kit is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with Mouse OPN antibody. OPN present in the sample is added and binds to antibodies coated on the wells. And then biotinylated Mouse OPN Antibody is added and binds to OPN in the sample. Then Streptavidin-HRP is added and binds to the Biotinylated OPN antibody. After incubation unbound Streptavidin-HRP is washed away during a washing step. Substrate solution is then added and color develops in proportion to the amount of Mouse OPN. The reaction is terminated by addition of acidic stop solution and absorbance is measured at 450 nm.
|Standard Solution (16ng/ml)||0.5ml x1|
|Pre-coated ELISA Plate||12 * 8 well strips x1|
|Standard Diluent||3ml x1|
|Stop Solution||6ml x1|
|Substrate Solution A||6ml x1|
|Substrate Solution B||6ml x1|
|Wash Buffer Concentrate (30x)||20ml x1|
|Biotinylated Mouse OPN Antibody||1ml x1|
|Plate Sealer||2 pics|
|Zipper bag||1 pic|
Material Required But Not Supplied
Serum Allow serum to clot for 10-20 minutes at room temperature. Centrifuge at 2000-3000 RPM for 20 minutes.
Plasma Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 2000-3000 RPM at 2 - 8°C within 30 minutes of collection.
Urine Collect by sterile tube. Centrifuge at 2000-3000 RPM for approximately 20 minutes. When collecting pleuroperitoneal fluid and cerebrospinal fluid, please follow the procedures above-mentioned.
Cell Culture Supernatant Collect by sterile tubes when examining secrete components. Centrifuge at 2000-3000 RPM for approximately 20 minutes. Collect the supernatants carefully. When examining the components within the cell, use PBS (pH 7.2-7.4) to dilute cell suspension to the cell concentration of approximately 1 million/ml. Damage cells through repeated freeze-thaw cycles to let out the inside components. Centrifuge at 2000-3000 RPM for approximately 20 minutes.
Tissue Rinse tissues in PBS (pH 7.4) to remove excess blood thoroughly and weigh before homogenization. Mince tissues and homogenize them in PBS (pH7.4) with a glass homogenizer on ice. Thaw at 2-8°C or freeze at -20°C. Centrifuge at 2000-3000 RPM for approximately 20 minutes.
All reagents should be brought to room temperature before use.
Standard Reconstitute the 120μl of the standard (16ng/ml) with 120μl of standard diluent to generate a 8ng/ml standard stock solution. Allow the standard to sit for 15 mins with gentle agitation prior to making dilutions. Prepare duplicate standard points by serially diluting the standard stock solution (8ng/ml) 1:2 with standard diluent to produce 4ng/ml, 2ng/ml, 1ng/ml and 0.5ng/ml solutions. Standard diluent serves as the zero standard(0 ng/ml). Any remaining solution should be frozen at -20℃ and used within one month. Dilution of standard solutions suggested are as follows:
|8ng/ml||Standard No.5||120μl Original Standard + 120μl Standard Diluent|
|4ng/ml||Standard No.4||120μl Standard No.5 + 120μl Standard Diluent|
|2ng/ml||Standard No.3||120μl Standard No.4 + 120μl Standard Diluent|
|1ng/ml||Standard No.2||120μl Standard No.3 + 120μl Standard Diluent|
|0.5ng/ml||Standard No.1||120μl Standard No.2 + 120μl Standard Diluen|
|Standard Concentration||Standard No.5||Standard No.4||Standard No.3||Standard No.2||Standard No.1|
Wash Buffer Dilute 20ml of Wash Buffer Concentrate 30x into deionized or distilled water to yield 500 ml of 1x Wash Buffer. If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved.
1. Prepare all reagents, samples and standards.
2. Add sample and ELISA reagent into each well. Incubate for 1 hour at 37°C.
3. Wash the plate 5 times.
4. Add substrate solution A and B. Incubate for 10 minutes at 37°C.
5. Add stop solution and color develops.
6. Read the OD value within 10 minutes.
Contact Person: Lee