Mouse TNF Alpha PicoKine™ ELISA Kit

SKU EK0527
Size 96wells/kit, with removable strips.
Reactivity Mouse
Sample Type cell culture supernates, cell lysates, serum and plasma (heparin, EDTA).
Sample Volume 100ul per well
Sensitivity <1pg/ml
Assay Range 15.6pg/ml-1000pg/ml(cellculturesupernates),7.8pg/ml-500pg/ml(mouseserum,plasma)

Overview

Product Name Mouse TNF Alpha PicoKine™ ELISA Kit
SKU/Catalog Number EK0527
Storage & Handling Store at 4°C for 6 months, at -20°C for 12 months. Avoid multiple freeze-thaw cycles(Shipped with wet ice.)
Size 96wells/kit, with removable strips.
Description Sandwich High Sensitivity ELISA kit for Quantitative Detection of Mouse TNF alpha. 96wells/kit, with removable strips.
Cite This Product Mouse TNF Alpha PicoKine™ ELISA Kit (Boster Biological Technology, Pleasanton CA, USA, Catalog # EK0527)
Sample Type cell culture supernates, cell lysates, serum and plasma (heparin, EDTA). . Anticoagulant(s): heparin or EDTA
*The recommended anticoagulants are proven to not block the antibody binding sites on the target antigen. Please do not collect blood sample with other anticoagulants that are not specified above or contact us to check for feasibility.
Sensitivity <1pg/ml
Assay Range 15.6pg/ml-1000pg/ml(cellculturesupernates),7.8pg/ml-500pg/ml(mouseserum,plasma)
Immunogen Expression system for standard: E.coli; Immunogen sequence: L80-L235
Reactivity Mouse
Cross Reactivity There is no detectable cross-reactivity with other relevant proteins.
Antibody Clonalities Capture Antibody | Detection Antibody:
monoclonal antibody from rat|polyclonal antibody from goat

Assay Details

Kit Components

Catalog number Description Quantity
EK0527-CAP 96-well plate precoated with anti-Mouse Tnf antibody 1
EK0527-ST lyophilized recombinant Mouse Tnf standard 10ng/tube
EK0527-DA biotinylated anti-Mouse Tnf antibody 130ul
AR1103 Avidin-Biotin-Peroxidase Complex(ABC) 130ul
AR1106-1 sample diluent buffer 30ml
AR1106-2 antibody diluent buffer 12ml
AR1106-3 ABC diluent buffer 12ml
AR1104 TMB color developing agent 10ml
AR1105 TMB stop solution 10ml
PLA-SEA Adhesive cover 4

*Why there is no wash buffer? Our Avidin-Biotin-Peroxidase Diluent contains the detergent (TWEEN) normally present in other companies' ELISA kits. This saves you the step of having to wash with the special wash buffer and achieve similar or better signal to noise ratio. The wash can use regular wash buffers (PBS, TBS etc.) commonly found in labs.

Materials Required But Not Provided

  • Microplate reader in standard size.
  • Automated plate washer.
  • Adjustable pipettes and pipette tips. Multichannel pipettes are recommended in the condition of large amount of samples in the detection.
  • Clean tubes and Eppendorf tubes.
  • Washing buffer (neutral PBS or TBS).
  • Preparation of 0.01M TBS: Add 1.2g Tris, 8.5g NaCl; 450μl of purified acetic acid or 700μl of concentrated hydrochloric acid to 1000ml H2
  • Preparation of 0.01 M PBS: Add 8.5g sodium chloride, 1.4g Na O and adjust pH to 7.2-7.

Typical Data Obtained from Mouse TNF Alpha PicoKine™ ELISA Kit

(TMB reaction incubate at 37°C for 15-25min)

Concentration(pg/ml)015.631.262.51252505001000
O.D.0.0280.0900.1560.2810.5430.9231.4842.258

Intra/Inter Assay Precision

Intra-Assay PrecisionInter-Assay Precision
Sample123123
n161616242424
Mean(pg/ml)18502991653305
Standard deviation0.823.922.120.914.3924.4
CV(%)4.6%7.8%7.4%5.7%8.3%8%

Reproducibility

Three samples with differing target protein concentrations were assayed using four different lots to measure the CV% lot to lot variance.

Reproducibility

To assay reproducibility, three samples with differing target protein concentrations were assayed using four different lots.

LotsLot1 (pg/ml)Lot2 (pg/ml)Lot3 (pg/ml)Lot4 (pg/ml)Mean (pg/ml)Standard DeviationCV (%)
Sample 118181719180.73.8%
Sample 250545447512.945.7%
Sample 32992942932852925.011.7%
*number of samples for each test n=16.

*The typical data is obtained from Boster's internal QC result and for reference only. It may differ from the lab test results of the end users. It is more important that the user's lab test results reflect the same linearity demonstrated in the typical data than achieving exactly the same O.D. values.

Target Info

Protein Target Info (Source: Uniprot.org)

Uniprot Id P06804
Gene Name Tnf
Protein Name Tumor necrosis factor
Alternative Names Tumor necrosis factor;Cachectin;TNF-alpha;Tumor necrosis factor ligand superfamily member 2;TNF-a;Tumor necrosis factor, membrane form;N-terminal fragment;NTF;Intracellular domain 1;ICD1;Intracellular domain 2;ICD2;C-domain 1;C-domain 2;Tumor necrosis factor, soluble form;Tnf;Tnfa, Tnfsf2;
Subcellular Localization Cell membrane; Single-pass type II membrane protein.
Molecular Weight 25896 MW

*if product is indicated to react with multiple species, protein info is based on the human gene.

Ontology

Protein Function Cytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation.
Research Areas Mouse

*You can search these to find other products in these research areas.
Background Tumor necrosis factor-alpha(TNF-alpha, or TNF) is secreted by macrophages in response to inflammation, infection and cancer. Human Tumor Necrosis Factor(TNF) and Lymphotoxin(TNF-beta) are cytotoxic proteins which have similar biological activities and share 30% amino acid homology. TNF-alpha is produced by monocytes, which can stimulate endothelial cells to produce the multilineage growth factor granulocyte-macrophage colony-stimulating factor and extend the role of this immunoregulatory protein to the regulation of hematopoiesis in vitro. TNF is a soluble protein that causes damage to tumor cells but has no effect on normal cells. Human TNF has been purified to apparent homogeneity as a 17.3-kilodalton protein from HL-60 leukemia cells and has showed cytotoxic and cytostatic activities against various human tumor cell lines. The human TNF cDNA is 1585 base pairs in length and encodes a protein of 233 amino acids. The mature protein begins at residue 77, leaving a long leader sequence of 76 amino acids. TNF-alpha has been mapped to human chromosome 6.

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Detect Mouse Tnf Alpha/TNF with <1pg/ml sensitivity. Format: 96-well plate with removable strips. Compatible samples: cell culture supernates, cell lysates, serum and plasma (heparin, EDTA). This is a TMB colorimetric sandwich ELISA kit with short assay time and fast experiment set up. Tnf Alpha/TNF tissue specificity:
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EK0527
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$369.00

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Customer Q&As

  • Q: There are precipitates in the sample diluent buffer. What is it?
    A: The precipitates are fatty substances contained in BSA. It would not affect the performance of the kit. If you want to confirm this, please contact Boster Support.
  • Q: I left this product out in ambient temperatures for around 5 days, will the kit be stable?
    A: Our kits should still be stable after being in ambient temperatures for around 5 days. It would not affect the performance of the kit. We suggest you to run the test and contact us if does not work as expected.
  • Q: What is the number of density of cells recommended for ELISA assay (for cell culture supernants and cell lysates)?
    A: The number of cells we recommened for cell culture supernants and cell lysates is 10^6 cells/mL.
  • Q: How many samples can I test on one ELISA plate?
    A: On each 96 well plate, we recommend running an 8-point standard curve with duplicate wells. With the remaining 80 wells, 40 samples can be tested in duplicate.
  • Q: The volumes of my samples are small, would it be possible to use 50µl/sample?
    A: There are two solutions for insufficient sample volume. <br> 1. Dilute the samples with the provided sample diluent buffer into 100ul. <br> 2. If 50ul of sample is added into a well, add 50ul of standard solution.
  • Q: Will this kit work on tissue samples?
    A: For tissue homogenates, endogenous biotin should be considered. Endogenous biotin in tissue homogenates might introduce false positive signal. Please run the test as described below to confirm if there is Endogenous biotin in the tissue lysate samples using the ELISA kit. <br> • Add tissue homogenates into the wells and then add ABC and TMB without adding any biotinylated detection antibody to see if any signal will be observed. <br> • If no signal is produced, then you can work on the tissue sample by using the kit.
  • Q: Is it possible to dilute 10X TBS stock that is 250mM Tris Base/Tris-Hcl, 27 mM KCl, and 1.37M NaCl to be used as the wash buffer?
    A: You can dilute the 10X TBS stock to 1XTBS and test the pH value of the 1xTBS. <br> The 1XTBS can be used if the pH value falls in the range of 7.2-7.6.
  • Q: Would your kits work normally with samples from Wistar Rat?
    A: Our kits would work normally with samples from Wistar Rat.
  • Q: How can I store the dissolved wash buffer for a longer period of time?
    A: You can prepare a concentrated wash buffer (25X) which can be stored at 4°C for 1-3 months.
  • Q: Will EK0527 react with human samples?
    A: EK0527 does not react with human samples.
  • Q: Does the TMB color developing agent contain H2O2?
    A: Yes, the TMB color developing agent contain H2O2.
  • Q: Does this ELISA kit contain any product produced in humans or primates? <br> Keywords: component, ingredient
    A: None of the components in our ELISA kits are produced in humans or primates.
  • Q: What is the volume of the recombinant protein control? What is its shelf life? <br>Keywords: expiration, storage, temperature, size
    A: The volume of the control is around 100pg. If unopened, the shelf life of the control is the same as the whole kit - "Store at 4˚C for 6 months, at -20˚C for 12 months." The reconstituted control can be stored at -20˚C for 2 days.
  • Q: Will it be okay to run the experiment if I accidentally used the wrong buffer (e.g. sample diluent buffer) for antibody dilution instead of the antibody diluent buffer? Keywords: dilution, replace, substitute, sample buffer
    A: Using the sample diluent buffer for antibody dilution instead of the antibody diluent buffer will negatively affect the test result to some extent. The values of both standard and sample might be lower than the normal values.
  • Q: The protocol says to use neutral PBS and provides a recipe. Could I use neutral PBS made with KCl or KH2PO4 (common constituents for most PBS recipes) instead? Keywords: protocol, alternative buffer, applications, reagent recipe
    A: Yes, it is ok to use common PBS recipes. We have tried many types of buffers with common constituents, and no significant difference was observed whatsoever.
  • Q: My plasma samples have been somewhat hemolyzed. Does plasma hemolysis have any effect on ELISA results? Keyword: application, protocol, troubleshooting
    A: There will be some unspecific staining in hemolysis samples and make the result inaccurate. We suggest you collect plasma samples again, hemolysis samples are not recommended to use.
  • Q: Why are my O.D. values different than your values on the datasheet? Keyword: troubleshooting, reference
    A: We detected the kit in our lab and got our values on the datasheet before delivery, but protein activity will decrease as time goes on, so you may get lower O.D. values than ours. However, it should still be in a reasonable range and the standards can be used to calculate sample values. Good linearity of curve is more important than the actual numerical value.
  • Q: On the ELISA kit datasheet it says "HRP substrate TMB was used to visualize HRP enzymatic reaction." What is HRP and which part of the kit contains HRP? Keyword: kit components, reagents, protocol
    A: It means Avidin-Biotin-Peroxidase Complex(AR1103) and you could find it in Kit Components.
  • Q: Do you offer any ELISA kits that can work with whole blood samples instead of plasma or urine? Keyword: applications
    A: We test serum and plasma routinely, and there is very little difference between serum, plasma and whole blood. The whole blood also contains proteins we need to test. The kit can be used to test whole blood in theory.
  • Q: Does silicic acid (formula SiOH4) affect the results of the ELISA assay? Keyword: protocol, reagents
    A: The acid may affect the binding of antigen to antibody, it is not recommended to use. That being said, it is unlikely to affect the reaction if the solution remains an overall neutral environment.
  • Q: Is there lot-to-lot variation of the ELISA kits? What are Boster's general services when I have questions about your kits? Keyword: technical support, help, customer service
    A: The NIBSC/WHO 1st International Standard is evaluated, we always test our kits before delivery, and customers can find the test result on the protocol. If the customer needs technical support from us to analyze their data, please contact us and we ask that you provide us the following information: the lot# and production date, and when did the customer detect the kit.
  • Q: The results of my standards do not look correct, what could be the problem? Keyword: troubleshoot, protocol
    A: Double check the protocol for plate washing. It should be examined on three aspects: 1) Did you make the washing buffer correctly? 2) How long did you let the buffer stay in wells? 3) What was the volume of the washing buffer added to each well? In addition, pay attention when adding sample to avoid contamination. And we suggest testing the standards again. Values of standards at low concentration are more affected than that at higher concentration, so customer can still get expected values at high concentration even if there is an error. If problems persist, please contact technical support with the catalog and lot number of your product.
  • Q: What is the well depth of the 96 well plates for the ELISA kits? Keyword: well capacity, product size
    A: The well depth is 300ul, and the max capacity is 350ul. The height of the well is 1.1 cm, and the internal size is 1 cm.
  • Q: The kit does not include wash solution, what should I do? Keyword: wash buffer
    A: Our Elisa kits do not come with wash solution, but we have included information about how to make washing buffer on the datasheet. Please refer to the "Material Required But Not Provided" section. We also offer washing buffer for sale separately (Phosphate Buffered Saline Powder SKU: AR0030).
  • Q: For your ELISA kits, are the capture and detection antibodies polyclonals or monoclonals? Keyword: antibody clonality
    A: This information can be found for each kit under the "Properties" section, and you can find the immunogen sequence information in the "Overview" section.
  • Q: Do your ELISA kits come with sealants or plate covers? Keyword: storage, sequential use
    A: The kit can be used within a month sequentially if it's opened and stored at 4 degree. There is no need to use sealants, the plate can be packaged with aluminum foil bag, and for other reagents keep bottle tightly closed.
  • Q: What is the concentration of Sodium Azide in your Elisa kits? Keyword: preservative
    A: Our Elisa kits contain 0.02% Sodium Azide. All of the components except TMB colour developing reagent and stop solution contains 0.02% Sodium azide as the preservative.
  • Q: Are there positive and negative controls available for my ELISA kit? Keyword: positive control, negative control
    A: We can provide a recombinant protein as a control. It costs $50 per control and takes 2 weeks to manufacture. We cannot provide a positive or negative control in serum.
  • Q: Why do I get positive results for my knockout (KO) model when used as a control?
    A: The knockout (KO) model may contain truncated forms of the target protein which can be detected by the ELISA assay.
  • Q: How long do I soak my plate in the wash buffer?
    A: The plate should soak in 0.3mL PBS or TBS wash buffer for at least 1-2 minutes in an automated wash station.
  • Q: Can I use Tween in my wash buffer?
    A: While it is not recommended to use Tween in your wash buffer, small amounts (<0.1% concentration) may decrease background due to insufficient blocking.
  • Q: What plate type do I use to set up the microplate reader?
    A: Our plates are made with the Corning costar plates similar to the DNA-BIND 96 -well plates.
  • Q: What should I use for negative control?
    A: Please contact us for negative control suggestions. You can also check expression databases such as genecards, uniprot etc. Due to logistic reasons, we do not sell serum or lysates that we use internally for positive or negative control.
  • Q: Where can I find troubleshooting information? What should I do if I have unexpected bands, high background, no signal, weak signal
    A: You can find Boster's troubleshoot guides under tech support tab. Please contact us for further assistance on troubleshooting your experiment.
  • Q: What is the normal level of this protein in my sample of interest?
    A: We have reviewed literature and have gathered this information for most of our ELISA kits. You can find this information on the product page or contact us if you cannot find it. However this information is only suggestive and cannot replace pilot studies in determining the optimal sample dilution ratio.
  • Q: Is the plate separable? Can I use only part of the kit and save the rest for later? How many samples can I run with one kit?
    A: Yes the plate is separable if the kit says that there are removable strips. There are 12 strips of 8 wells each, all removable from the plate. The amount of samples you can run depend on a few factors. In the most common ELISA set up, you will use two strips for standards, and 10 strips for samples using duplicates, which let you run up to 40 samples per kit. Contact us if you have questions regarding other situations.
  • Q: Does the kit contain sample preparation reagents? How do I prepare my samples?
    A: Since different sample types require different reagents, we do not include them in the ELISA kit. However we do have each reagent mentioned in the file below available at very reasonable prices. Be sure to check them out. For sample preparation protocols please download the file below: https://www.bosterbio.com/media/pdf/Boster_Sample_Preparation_Protocols.pdf
  • Q: Can this ELISA kit work on brain tissue homogenate, cell culture supernatant, saliva, urine, serum, whole blood or any other sample type?
    A: In theory the ELISA kit will work for all sample types if the target protein is present at a level that falls within the linear range of the ELISA kit detection range. We guarantee the kit to work on the sample types that we have tested. If you want dilution ratio suggestions on these sample types please contact our technical support. For sample types that we have not tested for, we suggest you run pilot experiments to decide the optimal sample dilution.
  • Q: Can this ELISA kit react with the pro-form of the target protein? Can this ELISA kit react with an isoform of the protein?
    A: In general, unless otherwise specified, the ELISA kit is pan-specific, meaning that it will react with all different forms of the target protein if they share the majority of the target protein's sequence. The capture and detection antibodies are reactive to the entire sequence of the standard protein. You can find the sequence information of the standard protein in the "immunogen" section. For more information about the specificity of the kit for your particular experiment, please contact our technical support.
  • Q: Can this ELISA kit react with human, mouse, rat or other species?
    A: If the kit is reactive to another commonly used species (human, mouse, and/or rat), we would have listed it as a separate product. If you want to check cross-reactivity to a species that is not included in the 3 species listed above, please contact our technical support for more information. As a rule of thumb, if the sequences are 90%+ identical, there is a high chance of cross-reactivity for your species of interest.
  • Q: What are some alternative names that could be used to describe this product?
    A: Some common names include but are not limited to mouse tnf alpha elisa kit, mouse tnf-alpha elisa kit, mouse tnf elisa kit, mouse tnf a elisa kit, mouse tnf-a elisa kit
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