Rhesus Monkey Tumor Necrosis Factor (TNF) ELISA Kit, Lot 21AO-144 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P055)

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

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

100 μL

Pre-coated

1.Bring all reagents and samples to room temperature (18 - 25°C) before use.
2.Sample dilution.
3.Assay Diluent B (Item E) should be diluted 5-fold with deionized or distilled water before use.
4.Preparation of standard.
5.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 Diluent B (Item E) into the vial to prepare a detection antibody concentrate.
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 10,000-fold with 1x Assay Diluent B (Item E).

1.Bring all reagents and samples to room temperature (18 - 25°C) before use.
2.Add 100 µl of each standard 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 the solution and wash 4 times with 1x Wash Solution.
4.Add 100 µl of 1x prepared biotinylated antibody to each well. Incubate for 1 hour at room temperature with gentle shaking.
5.Discard the solution. Repeat the wash as in step 3.
6.Add 100 µl of prepared Streptavidin solution 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 to each well. Incubate for 30 minutes at room temperature in the dark with gentle shaking.
9.Add 50 µl of Stop Solution to each well. Read at 450 nm immediately.

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.

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

Avoid repeated freeze-thaw cycles.

-20 °C

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.

6 months

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.

For Research Use only

Tumor Necrosis Factor Alpha (TNFa)

DIF; TNF-alpha; TNFA; TNFSF2; RATTNF; Tnfa; tnf; TNF-a; TNFalpha; Tnfsf1a; TNFa; cTNF; Tnf-alpha; tnfa-like; TNF-ALPHA; dif; tnfa; xtnf; tnfsf2; tnf-alpha; Cachectin; tumor necrosis factor; tumor necrosis factor b (TNF superfamily; member 2); tumor necrosis factor alpha; tumor necrosis factor a (TNF superfamily; member 2); TNF; Tnf; tnf; tnfb; tnf-alpha; LOC103694380; tnfa

715467

P48094

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

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.