IL-6 ELISA Kit

Catalog No. ABIN1979356

Product Details IL-6 ELISA Kit

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

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: Rat IL-6 ELISA Kit for cell and tissue lysate samples.

Sample Type: Cell Lysate, Tissue Lysate

Analytical Method: Quantitative

Specificity: The antibody pair provided in this kit recognizes rat IL-6.

Cross-Reactivity (Details): This ELISA kit shows no cross-reactivity with any of the cytokines tested (e.g., rat CINC-2, CINC-3, CNTF, Fractalkine, IL-1alpha, IL- 1beta, IL-4, IL-10, GM-CSF, IFN-gamma, Leptin, Lix, MCP-1, MIP-3alpha, beta-NGF, TIMP-1, VEGF).

Sensitivity: 15 pg/mL

Characteristics:

• 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

Components:

• 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

Material not included:

• 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

Application Details

Sample Volume: 100 μL

Plate: Pre-coated

Protocol:

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.

Reagent Preparation:

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. The Rat IL-6 ELISA Kit Protocol 3 4. Preparation of standard: Briefly spin the vial of Item C. Add 500 µL 1x Sample Diluent Buffer (Item D) into Item C vial to prepare a 10,000 pg/ml standard solution. Dissolve the powder thoroughly by a gentle mix. Pipette 300 µL 1x Sample Diluent Buffer into each tube. Use the 10,000 pg/ml standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. 1x Sample Diluent Buffer serves as the zero standard (0 pg/ml). 200 µL Standard (Item C) + 500 µL 200myl 200 µL 200 µL 200 µL 200 µL 10,000 4,000 1,600 640 256 102.4 40.96 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) before use. HRP-Streptavidin concentrate should be diluted 400-fold with 1x Assay Diuent. The Rat IL-6 ELISA Kit Protocol 4 For example: Briefly spin the vial (Item G) and pipette up and down to mix gently . Add 30 µL of HRP-Streptavidin concentrate into a tube with 12 mL 1x Assay Diluent to prepare a 400-fold diluted HRP- Streptavidin solution (don't store the diluted solution for next day use). Mix well. 8. Cell lysate buffer should be diluted 2-fold with deionized or distilled water (for cell lysate and tissue lysate).

Assay Procedure:

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. 3. Discard 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 3. 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.

Calculation of Results:

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.

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

Restrictions: For Research Use only

Handling

Handling Advice: Avoid repeated freeze-thaw cycles.

Storage: -20 °C

Storage Comment: 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.

Expiry Date: 6 months

Target Details for IL-6

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

Alternative Name: IL-6 (IL6 Products)

Synonyms: BSF2 ELISA Kit, HGF ELISA Kit, HSF ELISA Kit, IFNB2 ELISA Kit, IL-6 ELISA Kit, Il-6 ELISA Kit, ILg6 ELISA Kit, Ifnb2 ELISA Kit, il6 ELISA Kit, CHIL-6 ELISA Kit, interleukin 6 ELISA Kit, interleukin-6 ELISA Kit, IL6 ELISA Kit, Il6 ELISA Kit, il-6 ELISA Kit, IL-6 ELISA Kit

Background: Interleukin-6 (IL-6)

Gene ID: 24498

UniProt: P20607

Pathways: TLR Signaling, 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, Autophagy, Cell RedoxHomeostasis, Cancer Immune Checkpoints, Inflammasome