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제품 설명
제품 번호
Cat. E-EL-H0101
제품 특징
●Sensitivity: 18.75 pg/mL.
●Detection Range: 31.25-2000 pg/mL
●Specificity: This kit recognizes Human IL-4 in samples. No significant cross-reactivity or interference between Human IL-4 and analogues was observed.
●Repeatability: Coefficient of variation is < 10%.
Sample collection
Serum: Allow samples to clot for 2 hours at room temperature or overnight at 4℃ before centrifugation for 15 min at 1000×g at 2~8℃. Collect the supernatant to carry out the assay. Blood collection tubes should be disposable and be non-endotoxin.
Plasma: Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 min at 1000×g at 2~8℃ within 30 min of collection. Collect the supernatant to carry out the assay. Hemolysed samples are not suitable for ELISA assay!
Cell lysates: For adherent cells, gently wash the cells with moderate amount of pre-cooled PBS and dissociate the cells using trypsin. Collect the cell suspension into a centrifuge tube and centrifuge for 5 min at 1000×g. Discard the medium and wash the cells 3 times with pre-cooled PBS. For each 1×106 cells, add 150-250 μL of pre-cooled PBS to keep the cells suspended. Repeat the freeze-thaw process several times until the cells are fully lysed. Centrifuge for 10min at 1500×g at 4℃. Remove the cell fragments, collect the supernatant to carry out the assay. Avoid repeated freeze-thaw cycles.
Tissue homogenates: It is recommended to get detailed references from the literature before analyzing different tissue types. For general information, hemolysed blood may affect the results, so the tissues should be minced into small pieces and rinsed in ice-cold PBS (0.01M, pH=7.4) to remove excess blood thoroughly. Tissue pieces should be weighed and then homogenized in PBS (tissue weight (g): PBS (mL) volume=1:9) with a glass homogenizer on ice. To further break down the cells, you can sonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5 min at 5000×g to get the supernatant.
Cell culture supernatant or other biological fluids: Centrifuge samples for 20 min at 1000×g at 2~ 8℃. Collect the supernatant to carry out the assay.
시약 준비 과정
1. Bring all reagents to room temperature (18~25℃) before use. Follow the Microplate reader manual for set-up and preheat it for 15 min before OD measurement.
2. Wash Buffer: Dilute 30 mL of Concentrated Wash Buffer with 720 mL of deionized or distilled water to prepare 750 mL of Wash Buffer. Note: if crystals have formed in the concentrate, warm it in a 40℃ water bath and mix it gently until the crystals have completely dissolved.
3. Standard working solution: Centrifuge the standard at 10,000×g for 1 min. Add 1.0 mL of Reference Standard &Sample Diluent, let it stand for 10 min and invert it gently several times. After it dissolves fully, mix it thoroughly with a pipette. This reconstitution produces a working solution of 2000pg/mL. Then make serial dilutions as needed. The recommended dilution gradient is as follows: 2000、1000、 500、250、125、62.5、31.25、0 pg/mL. Dilution method: Take 7 EP tubes, add 500uL of Reference Standard & Sample Diluent to each tube. Pipette 500uL of the 2000pg/mL working solution to the first tube and mix up to produce a 1000pg/mL working solution. Pipette 500uL of the solution from the former tube into the latter one according to this step. The illustration below is for reference. Note: the last tube is regarded as a blank. Don’t pipette solution into it from the former tube.
4. Biotinylated Detection Ab working solution: Calculate the required amount before the experiment (100 μL/well). In preparation, slightly more than calculated should be prepared. Centrifuge the stock tube before use, dilute the 100× Concentrated Biotinylated Detection Ab to 1×working solution with Biotinylated Detection Ab Diluent.
5. Concentrated HRP Conjugate working solution: Calculate the required amount before the experiment (100 μL/well). In preparation, slightly more than calculated should be prepared. Dilute the 100× Concentrated HRP Conjugate to 1× working solution with Concentrated HRP Conjugate Diluent.
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