TNF alpha ELISA Kit

Catalog No. ABIN7672435

The Rat TNF alpha ELISA Kit (ABIN7672435) is a Colorimetric ELISA Kit designed to quantify Rat TNF alpha.

Quick Overview for TNF alpha ELISA Kit (ABIN7672435)

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Reactivity: Rat

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 3.906 pg/mL - 250 pg/mL

Application: ELISA

Sample Type: Biological Fluids, Cell Culture Supernatant, Cell Lysate, Plasma, Serum, Tissue Lysate

Grade: Quick

Product Details TNF alpha ELISA Kit

Minimum Detection Limit: 3.906 pg/mL

Purpose: Rat TNF-α (Tumor Necrosis Factor Alpha) QuickTest ELISA Kit

Analytical Method: Quantitative

Specificity: Specifically binds with TNF-α , no obvious cross reaction with other analogues. Recommended Sample Dilution Ratio The following table shows the recommended dilution ratios for this kit for a limited number of samples for your reference only. (When the sample content is very low, the original solution can be added without dilution, but it is necessary to ensure that the pH is between 6.8 and 8.0, and it does not contain more than 10 % organic solvents or high-concentration protein denaturants.) 1. Common sample validation: Sample Type Recommended Dilution Ratio Content Normal Rat serum (n=15) undiluted ND-25pg/mL Normal Rat plasma (EDTA, Citrate , heparin) (n=15) undiluted ND-30pg/mL Rat spleen cells were cultured with 5 %FBS + 1640 + double antibody for 48 hours undiluted 13pg/mL Rat spleen cells were cultured with 5 %FBS + 1640 + double antibody + 100 ng/mL LPS for 48 hours 1/10 dilution 905pg/mL Rat spleen cells were cultured with 5 %FBS+ 1640 + double antibody +100 ng/mL LPS for 48 hours. After that, 300 ng/mL Brefeldin A (BFA) was added and cultured for 3 hours. Add cell lysis buffer(Catalogue No.:E050), collect the lysate solution (total protein concentration measured by BCA assay: 2.1 mg/mL) to detect. 1/20 dilution 996pg/mg(total protein) 2. KD sample validation (Detect TNF-α-KD NR8383 cells): Sample Type Dilution Ratio Content Wild NR8383 cells were stimulated with 1 μg/mL LPS for 12 hours to detect cell culture supernatant 1/2-1/5 dilution 635pg/mL NR8383 cells (+siRNA) were stimulated with 1 μg/mL LPS for 12 hours to detect cell culture supernatants undiluted ND NR8383 cells (+siRNA) were treated with 1 μg/mL LPS for 12 hours and then cultured with 300 ng/mL Brefeldin A (BFA) for 3 hours. Add cell lysis buffer(Catalogue No.:E050), collect the lysate solution (total protein concentration measured by BCA assay: 1.28 mg/mL) to detect. undiluted 8pg/mg(total protein) Note:ND is lower than the sensitivity of the kit and was not detected 3. Antibody by WB KD validation (Detect TNF-α-KO NR8383 cells):

Sensitivity: 2.344 pg/mL

Components:

• ELISA Microplate(Dismountable)
• Lyophilized Standard
• Cap/Det Ab (Ready to use
• blue)
• TMB Substrate
• Sample Dilution Buffer
• Stop Solution
• Plate Sealer
• Product Description

Material not included:

• Microplate reader (wavelength: 450nm)
• 37 °C incubator (CO2 incubator for cell culture is not recommenced.)
• Automated plate washer or multi-channel pipette/5 mL pipettor
• Precision single (0.5-10μL
• 5-50μL
• 20-200μL
• 200-1000μL) and multi-channel pipette with disposable tips(Calibration is required before use.)
• Sterile tubes and Eppendorf tubes with disposable tips
• Absorbent paper and loading slot
• Deionized or distilled water

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

Sandwich ELISA, Double Antibody

Sample Volume: 50 μL

Assay Time: 2 h

Plate: Pre-coated

Protocol: Step 1: Take out the required plate wells, add 50 µL Cap/Det Ab into each well, then add 50 µL Standard or Sample into individual well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure thorough mixing then static incubate for 60 minutes at 37 °C. Washing: Wash the plate twice without immersion. Step 2: Add 100 µL HRP-Streptavidin (orange) into each well, seal the plate and static incubate for 30 minutes at 37 °C. Washing: Wash the plate five times without immersion. Step 3: Add 90 µL TMB substrate solution, seal the plate and static incubate for 10-20 minutes at 37 °C. (Accurate TMB visualization control is required.) Step 4: Add 50 µL stop solution. Read at 450nm immediately and calculate.

Assay Procedure:

1. Take out the required strips, add 50 µL Cap/Det Ab into each well, then add 50 µL Standard or Sample into relevant well. (When adding standard or sample, the disposable tip lightly touches the liquid level. Change the disposable tips for different samples and standards.) Gently tap the plate for 10s to ensure complete mixing then statically incubate for 60 minutes at 37 °C.
2. Wash the plate twice without immersion.
3. Add 100 µL HRP-Streptavidin (orange) into each well, seal the plate and statically incubate for 30 minutes at 37 °C.
4. Wash the plate five times without immersion.
5. Add 90 µL TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37 °C (Accurate TMB visualization control is required.).
6. Add 50 µL stop solution. Read at 450nm immediately and calculate.

Restrictions: For Research Use only

Handling

Handling Advice: Avoid direct light exposure to antibody solutions

Storage: 4 °C

Storage Comment: Store sealed Kit at 2-8° C. Do Not Cryopreserve. Store ELISA Microplate for 1 month at 2-8°C, Store for 12 month at -20°C. Put the leftover strips into a sealed foil bag with the desiccant

Expiry Date: 12 months

Target Details for TNF alpha

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Alternative Name: TNF-alpha

Background: TNF-α, DIF, TNF-alpha, TNFA, TNFSF2

UniProt: P16599

Pathways: NF-kappaB Signaling, Apoptosis, Caspase Cascade in Apoptosis, TLR Signaling, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome