IL-6 ELISA Kit

Produktnummer ABIN625151

Dieses Colorimetric ELISA-Kit wurde entwickelt für die quantitative Messung von Maus IL-6. Es ist 1 Publikation verfügbar.

Kurzübersicht für IL-6 ELISA Kit (ABIN625151)

Target: IL-6 (IL6) (Interleukin 6 (IL6))

Reaktivität: Maus

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Proben: Cell Lysate, Tissue Lysate

Produktdetails IL-6 ELISA Kit

Verwendungszweck: Mouse IL-6 ELISA Kit for cell and tissue lysate samples.

Analytische Methode: Quantitative

Spezifität: The antibody pair provided in this kit recognizes mouse IL-6.

Sensitivität: 2 pg/mL

Produktmerkmale:

• Strip plates and additional reagents allow for use in multiple experiments
• Quantitative protein detection
• Establishes normal range
• The best products for confirmation of antibody array data

Bestandteile:

• Pre-Coated 96-well Strip Microplate
• Wash Buffer
• Stop Solution
• Assay Diluent(s)
• Lyophilized Standard
• Biotinylated Detection Antibody
• Streptavidin-Conjugated HRP
• TMB One-Step Substrate

Benötigtes Material:

• Distilled or deionized water
• Precision pipettes to deliver 2 μL to 1 μL volumes
• Adjustable 1-25 μL pipettes for reagent preparation
• 100 μL and 1 liter graduated cylinders
• Tubes to prepare standard and sample dilutions
• Absorbent paper
• Microplate reader capable of measuring absorbance at 450nm
• Log-log graph paper or computer and software for ELISA data analysis
• Cell lysate buffer

Anwendungsinformationen

Probenmenge: 100 μL

Plattentyp: Pre-coated

Protokoll:

1. Prepare all reagents, samples and standards as instructed in the manual.
2. Add 100 μL of standard or sample to each well.
3. Incubate 2.5 h at RT or O/N at 4 °C.
4. Add 100 μL of prepared biotin antibody to each well.
5. Incubate 1 h at RT.
6. Add 100 μL of prepared Streptavidin solution to each well.
7. Incubate 45 min at RT.
8. Add 100 μL of TMB One-Step Substrate Reagent to each well.
9. Incubate 30 min at RT.
10. Add 50 μL of Stop Solution to each well.
11. Read at 450 nm immediately.

Aufbereitung der Reagenzien:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use.
   2. Sample dilution: Tissue lysate and cell lysate sample should be diluted at least 5-fold with 1x Sample Diluent Buffer.
   3. Sample Diluent Buffer (Item D) and Assay Diluent (Item E) should be diluted 5-fold with deionized or distilled water before use.
   4. Preparation of standard: Briefly spin the vial of Item C. Add 640 µL 1x Sample Diluent Buffer (Item D) into Item C vial to prepare a 50 ng/mL standard. Dissolve the powder thoroughly by a gentle mix. Add 8myl IL-6 standard from the vial of Item C, into a tube with 658.8 µL Sample Diluent Buffer to prepare a 600 pg/mL stock standard solution. Pipette 400 µL 1x Sample Diluent Buffer into each tube. Use the stock standard solution to produce a dilution series . Mix each tube thoroughly before the next transfer. 1x Sample Diluent Buffer serves as the zero standard (0 pg/mL). 200 µL 200myl 8 µL standard + 658.7myl 200 µL 200 µL 200 µL 600 200 66.7 22.2 7.4 2.5 0.82 0 pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL pg/mL
   5. If the Wash Concentrate (20x) (Item B) contains visible crystals, warm to room temperature and mix gently until dissolved. Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer.
   6. Briefly spin the Detection Antibody vial (Item F) before use. Add 100 µL of 1x Assay Diuent into the vial to prepare a detection antibody concentrate. Pipette up and down to mix gently (the concentrate can be stored at 4 °C for 5 days). The detection antibody concentrate should be diluted 80-fold with 1x Assay Diuent and used in step 4 of Part VI Assay Procedure.
   7. Briefly spin the HRP-Streptavidin concentrate vial (Item G) and pipette up and down to mix gently before use. HRP-Streptavidin concentrate should be diluted 200-fold with 1x Assay Diuent. For example: add 50 µL of HRP-Streptavidin concentrate into a tube with 10 ml 1x Assay Diluent to prepare a 200 fold diluted HRP- Streptavidin solution. Mix well.
   8. Cell lysate buffer should be diluted 2-fold with deionized or distilled water (for cell lysate and tissue lysate).

Testdurchführung:

1. Bring all reagents and samples to room temperature (18 - 25 °C) before use. It is recommended that all standards and samples be run at least in duplicate.
   2. Add 100 µL of each standard (see Reagent Preparation step 2) and sample into appropriate wells. Cover well and incubate for 2.5 hours at room temperature or over night at 4 °C with gentle shaking. We recommend using 50-500 myg/mL of total protein for lysate sample. The amount of sample used depends on the abundance of target protein. More of the sample can be used if signals are too weak. If signals are too strong, the sample can be diluted further.
   3. Discard the solution and wash 4 times with 1x Wash Solution. Wash by filling each well with Wash Buffer (300 myl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
   4. Add 100 µL of 1x prepared biotinylated antibody (Reagent Preparation step 6) to each well. Incubate for 1 hour at room temperature with gentle shaking.
   5. Discard the solution. Repeat the wash as in step
   6. Add 100 µL of prepared Streptavidin solution (see Reagent Preparation step 7) to each well. Incubate for 45 minutes at room temperature with gentle shaking.
   7. Discard the solution. Repeat the wash as in step
   8. Add 100 µL of TMB One-Step Substrate Reagent (Item H) to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
   9. Add 50 µL of Stop Solution (Item I) to each well. Read at 450 nm immediately.

Ergebnisberechnung:

Calculate the mean absorbance for each set of duplicate standards, controls and samples, and subtract the average zero standard optical density. Plot the standard curve on log-log graph paper or using Sigma plot software, with standard concentration on the x-axis and absorbance on the y-axis. Draw the best-fit straight line through the standard points.
Typical Data: These standard curves are for demonstration only. A standard curve must be run with each assay. Sample Dilunt Buffer Mouse IL-6 concentration (pg/mL) 0.1 1 10 100 1000 O D =4 50 n m 0.01 0.1 1 10
Sensitivity: The minimum detectable dose of IL-6 is typically less than 2 pg/mL.
Recovery: Recovery was determined by spiking various levels of mouse IL-6 into mouse tissue lysate and cell lysate. Mean recoveries are as follows: Sample Type Average % Recovery Range ( %) Tissue lysate 93.39 83-103 Cell lysate 94.53 84-104
Linearity: Sample Type Cell lysate Tissue lysate 1:2 Average % of Expected 94 96 Range ( %) 83-103 84-105 1:4 Average % of Expected 93 92 Range ( %) 84-103 83-104
Reproducibility: Intra-Assay: CV<10 % Inter-Assay: CV<12 %

Testpräzision: Intra-Assay: CV< 10 % Inter-Assay: CV< 12 %

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Handhabung: Avoid repeated freeze-thaw cycles.

Lagerung: -20 °C

Informationen zur Lagerung: The entire kit may be stored at -20°C for up to 1 year from the date of shipment. Avoid repeated freeze-thaw cycles. The kit may be stored at 4°C for up to 6 months. For extended storage, it is recommended to store at -80°C.

Haltbarkeit: 6 months

Referenzen für IL-6 ELISA Kit (ABIN625151)

Wu, Zhao, Chen, Cheng, Zhang: "Galantamine attenuates amyloid-β deposition and astrocyte activation in APP/PS1 transgenic mice." in: Experimental gerontology, Vol. 72, pp. 244-50, (2015) (PubMed).

Details zu IL-6

Target: IL-6 (IL6) (Interleukin 6 (IL6))

Andere Bezeichnung: IL-6

Hintergrund: The Mouse IL-6 ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of mouse IL-6 cell lysate and tissue lysate. This assay employs an antibody specific for mouse IL-6 coated on a 96-well plate. Standards and samples are pipetted into the wells and IL-6 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-mouse IL-6 antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of IL-6 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm. Reproducibility: Intra-Assay: CV<10% Inter-Assay: CV<12%.

Gen-ID: 16193

UniProt: P08505

Pathways: TLR Signalweg, Hormone Transport, Negative Regulation of Hormone Secretion, Myometrial Relaxation and Contraction, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Regulation of Carbohydrate Metabolic Process, Autophagie, Cell RedoxHomeostasis, Cancer Immune Checkpoints, Inflammasome