This kit is based on Double antibody-Sandwich ELISA detection method and takes 4h assay time. The microplate provided in this kit has been precoated with anti TNF-α antibody. Add standard and properly diluted sample into relevant well respectively. After incubation, wash unbound components. Add biotinylated detection antibody. Then, it binds with TNF-α bound to precoated antibody. Wash unbound components and add HRP-Streptavidin Conjugate (SABC). Wash unbound components again and add TMB substrate solution. Then, TMB was catalyzed by HRP to produce a blue color product that turned yellow after adding a stop solution. Read the O.D. absorbance at 450nm in a microplate reader. Calculate the concentration of TNF-α in the sample by plotting standard curve. The concentration of the target substance is proportional to the OD450 value.
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- Product Name
- Mouse TNF-α(High sensitive Tumor Necrosis Factor Alpha)ELISA Kit
- Alias
- Tumor necrosis factor ELISA Kit, Cachectin ELISA Kit, TNF-alpha ELISA Kit, Tumor necrosis factor ligand superfamily member 2 ELISA Kit, TNF-a ELISA Kit, Tumor necrosis factor, membrane form ELISA Kit, N-terminal fragment ELISA Kit, NTF ELISA Kit, Intracellular domain 1 ELISA Kit, ICD1 ELISA Kit, Intracellular domain 2 ELISA Kit, ICD2 ELISA Kit, C-domain 1 ELISA Kit, C-domain 2 ELISA Kit, Tumor necrosis factor, soluble form ELISA Kit, TNF ELISA Kit, TNFA ELISA Kit, TNFSF2 ELISA Kit
- Catalogue No.
- EM0183-HS
- Size
- 48T/96T
- Species
- Mouse
- UniProt ID
- P06804
- Sample Type
- Serum, Plasma, Cell Culture Supernatant, cell or tissue lysate, Other liquid samples
- Detection Method
- Sandwich ELISA, Double Antibody
- Detection Wavelength
- OD450
- Reaction Duration
- 4 hours
- Range
- 1.563-100pg/ml (1pg=270mIU)
- Sensitivity
- 0.938pg/ml
- Storage
- 2-8°C(Sealed), Don't cryopreserve.
- 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. (The matrix components in serum/plasma will affect the test results, which it need to be diluted at least 1/2 with Sample Dilution Buffer before testing! When the content of other samples 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 mouse serum (n=16) 1/2 dilution ND-25pg/ml Normal mouse plasma (EDTA, Citrate , heparin) (n=16) 1/2 dilution ND-31pg/ml RAW 264.7 cells were cultured with 5%FBS + 1640 + double antibody for 12 hours 1/2dilution 28pg/ml RAW 264.7 cells were cultured with 5%FBS+ 1640 + double antibody +1μg/ml LPS, and the cell culture supernatant was detected after 12 hours 1/5-1/20 dilution 618pg/ml RAW 264.7 cells were cultured with 5%FBS+ 1640 + double antibody +1μg/ml LPS for 12 hours. After that, 300ng/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: 2.39 mg/ml) to detect. 1/50 dilution 1.31ng/mg(total protein) 2. KD sample validation (Detect TNF-α-KD RAW 264.7 cells):
Sample Type Dilution Ratio Content Wild RAW 264.7 cells were stimulated with 1μg/ml LPS for 12 hours to detect cell culture supernatant 1/20 dilution 711pg/ml RAW 264.7 cells (+siRNA) were stimulated with 1μg/ml LPS for 12 hours to detect cell culture supernatants 1/2 dilution ND RAW 264.7 cells (+siRNA) were treated with 1μg/ml LPS for 12 hours and then cultured with 300ng/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: 1.75 mg/ml) to detect. 1/2 dilution 17pg/mg(total protein) Note：ND is lower than the sensitivity of the kit and was not detected
3. 3. Antibody by WB KD validation (Detect TNF-α-KO RAW 264.7 cells):
Lane 1: Wild RAW 264.7 cells were treated with 1μg/ml LPS for 12 hours, then cultured with 300ng/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: 1.68 mg/ml with 10ug total protein loading) to detect.
Lane 2: RAW 264.7 cells (+siRNA) were treated with 1μg/ml LPS for 12 hours, then cultured with 300ng/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: 1.25 mg/ml with 10ug total protein loading) to detect. (no obvious band)
Lane 3.Wild RAW 264.7 cells were cultured for 12 hours (unstimulated), 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: 2.23 mg/ml with 10ug total protein loading) to detect.(no obvious band)
Primary antibody：
All lanes ：Capture rabbit monoclonal antibody 1ug/ml
Secondary antibody:
All lanes : HRP-Goat Anti-rabbit IgG (H) (Catalogue No.：FNSA-0004) at 1/5000 dilution
Molecular weight: 27 kDa
siRNA Target Sequences for Gene Knockdown:
Sense strand：5′-GACAACCAACUAGUGGUGC-3′
Antisense strand：5′-GCACCACUAGUUGGUUGUC-3′4. Verified recombinant protein:
(It is a normal phenomenon that some proteins have weak detection signals or cannot be detected at all due to differences in tags, sequences or protein activities)
A. HEK293-derived mouse TNF-alpha protein Leu80-Leu235
B. E. coli-derived mouse TNF-alpha protein Leu80-Leu235 - ELISA Kit Components
Kit Components Item Size(48T) Size(96T) Storage Condition for Opened Kit E001 ELISA Microplate(Dismountable) 8×6 8×12 Put the rest strips into a sealed foil bag with the desiccant. Stored for 1 month at 2-8°C; Stored for 12 month at -20°C E002 Lyophilized Standard 1vial 2vial Put the rest standards into a desiccant bag. Stored for 1 month at 2-8°C; Stored for 12 month at -20°C E003 Biotin-labeled Antibody(Concentrated, 100X) 60ul 120ul 2-8°C (Avoid Direct Light) E034 HRP-Streptavidin Conjugate(SABC, 100X) 60ul 120ul E024 TMB Substrate 5ml 10ml E039 Sample Dilution Buffer 10ml 20ml 2-8°C E040 Antibody Dilution Buffer 5ml 10ml E049 SABC Dilution Buffer 5ml 10ml E026 Stop Solution 5ml 10ml E038 Wash Buffer(Concentrated, 25X) 15ml 30ml E006 Plate Sealer 3 pieces 5 pieces E007 Product Description 1 copy 1 copy - Required Instruments and Reagents
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- Microplate reader (wavelength: 450nm)
- 37°C incubator (CO2 incubator for cell culture is not recommenced.)
- Automated plate washer or multi-channel pipette/5ml pipettor (for manual washing purpose)
- 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
- Assay Procedure Summary
-
- Step 1: Add 100ul standard or sample into each well, seal the plate and statically incubate for 90 minutes at 37°C.
- Washing: Wash the plate twice without immersing.
- Step 2: Add 100ul biotin-antibody working solution, seal the plate and statically incubate for 60 minutes at 37°C.
- Washing: Wash the plate three times and immerse for 1min each time.
- Step 3: Add 100ul HRP-Streptavidin Conjugate (SABC) working solution, seal the plate and statically incubate for 30 minutes at 37°C.
- Washing: Wash the plate five times and immerse for 1min each time.
- Step 4: Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
- Step 5: Add 50ul stop solution. Read at 450nm immediately and calculate.
- Standard Curve
-
This product is detected by QC department and meets performance required in the manual. (Laboratory Humidity: 20%-60%; Temperature: 18°C -25°C; Equilibrate TMB substrate to 37°C before staining. After adding into the ELISA wells, incubate for 15min at 37°C in dark.)
Due to different assay environments and operations, assay data below and standard curve are provided for reference. Experimenters should establish standard curve according to their own assay.
STD.(pg/ml) OD-1 OD-2 Average 0 0.095 0.099 0.097 1.562 0.185 0.194 0.189 3.125 0.307 0.323 0.314 6.25 0.51 0.536 0.521 12.5 0.73 0.767 0.745 25 1.209 1.271 1.234 50 1.718 1.806 1.754 100 2.07 2.176 2.113 
- Recovery
-
Add a certain amount of TNF-α into the sample. Calculate the recovery by comparing the measured value with the expected amount of TNF-α in the sample.
Sample Type Recovery Range(%) Average(%) serum(n=10) 87-104 97 EDTA plasma(n=10) 85-103 94 Heparin plasma(n=10) 86-105 96 - Linearity
-
Dilute the sample with a certain amount of TNF-α at 1:2, 1:4 and 1:8 to get the recovery range.
Sample Type 1:2 1:4 1:8 serum(n=10) 89-105% 88-104% 85-102% EDTA plasma(n=10) 85-101% 84-101% 84-101% Heparin plasma(n=10) 88-98% 92-95% 80-100% - Precision(%)
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Intra-assay Precision: samples with low, medium and high concentration are tested 20 times on the same plate.
Inter-assay Precision: samples with low, medium and high concentration are tested 20 times on three different plates.
Item Intra-assay Precision Inter-assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 Mean (pg/ml) 2.91 13.45 50.07 3.16 13.81 51.38 Standard deviation 0.14 0.67 3.21 0.13 0.58 3.04 CV(%) 4.95 4.95 6.42 4.23 4.23 5.91 - Stability
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Perform the stability test for the sealed kit at 37°C and 2-8°C and get relevant data.
ELISA kit(n=5) 37°C for 1 month 2-8°C for 6 months 2-8°C for 12 months Average(%) 80 95-100 85-98