小鼠1,4,5-三磷酸肌醇(IP3)ELISA kit#Mouse　inositol 1,4,5,-

 

This immunoassay kit allows for the in vitro quantitative determination of mouse inositol 1,4,5,-trisphosphate,IP3 concentrati*** in serum, pla***a, tissue homogenates and other biological fluids.

Inositol trisphosphate or inositol 1,4,5-trisphosphate (also commonly known as triphosphoinositol; abbreviated InsP3 or IP3), together with diacylglycerol, is a secondary messenger molecule used in signal transduction in biological cells. It is made by hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid that is located in the pla***a membrane, by phospholipase C.

IP3 binds to and activates the InsP3 receptor on the membrane of the endopla***ic reticulum (ER), and sarcopla***ic reticulum (SR) opens a calcium channel, resulting in the release of Ca2+ into the cytopla***, and sarcopla*** respectively. This increase in Ca2+ activates the ryanodine receptor-operated channel on the SR, leading to a further increase in the Ca2+.

 

The microtiter plate provided in this kit has been pre-coated with an antibody specific to IP3. Standa*** or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for IP3 and ***idin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain IP3, biotin-conjugated antibody and enzyme-conjugated ***idin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a w***elength of 450 nm &plu***n; 2 nm. The concentration of IP3 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

 

Reagent                                              Quantity

Assay plate                                                                    1 × 20ml

Standard                                             2

Sample Diluent                                                                    1 × 20ml

Assay Diluent A                                                                   1 × 10ml

Assay Diluent B                                                                  1 × 10ml

Detection Reagent A                                                           1 × 120μl

Detection Reagent B                                                         1 × 120μl

Wash Buffer(25 x concentrate)                          1 × 30ml

Substrate                                                                         1 × 10ml

Stop Solution                                                                             1 × 10ml

Plate sealer for 96 wells                                                      5

Instruction                                                                  1

Luminometer.

Pipettes and pipette tips.

EP tube

Deionized or distilled water.

Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20℃ or -80℃.

Pla***a - Collect pla***a using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8℃ within 30 minutes of collection. Store samples at -20℃ or -80℃. ***oid repeated freeze-thaw cycles.

Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20° C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20° C.

Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20℃ or -80℃. ***oid repeated freeze-thaw cycles.

Note: Serum, pla***a and tissue homogenates to be used within 7 days may be stored at 2-8 ℃, otherwise samples must stored at -20℃ (≤ 1 months) or -80℃ (≤ 2 months) to ***oid loss of bioactivity and contamination. ***oid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

 

1.  The kit should not be used beyond the expiration date on the kit label.

2.    Do not mix or substitute reagents with those from other lots or sources.

3.    If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.

4.    This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors h***e been tested in the Quantikine Immunoassay, the possibility of interference cannot be excluded.