Overview

  • Product name

    Rat TNF alpha ELISA Kit
    See all TNF alpha kits
  • Detection method

    Colorimetric
  • Sample type

    Cell culture supernatant, Cell culture extracts, Tissue Extracts
  • Assay type

    Sandwich (quantitative)
  • Sensitivity

    < 25 pg/ml
  • Range

    82.3 pg/ml - 20000 pg/ml
  • Recovery

    92 %

    Sample specific recovery
    Sample type Average % Range
    Cell culture extracts 93.17 81% - 105%
    Tissue Extracts 92.48 80% - 104%

  • Assay duration

    Multiple steps standard assay
  • Species reactivity

    Reacts with: Rat
  • Product overview

    Abcam’s TNF alpha ELSA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Rat TNF alpha in cell lysates and tissue lysates.

    This assay employs an antibody specific for Rat TNF alpha coated on a 96-well plate. Standards and samples are pipetted into the wells and TNF alpha present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Rat TNF alpha antibody is added. After washing away unbound biotinylated antibody, HRPconjugated streptavidin is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of TNF alpha bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

  • Tested applications

    Suitable for: Sandwich ELISAmore details
  • Platform

    Microplate

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 1 x 96 tests
    200X HRP-Streptavidin Concentrate 1 x 200µl
    20X Wash Buffer Concentrate 1 x 25ml
    2X Cell Lysis Buffer 1 x 5ml
    5X Assay Diluent 1 x 15ml
    5X Sample Diluent Buffer 1 x 10ml
    Biotinylated anti-Rat TNF alpha  (lyophilized)  2 vials
    Recombinant Rat TNF alpha Standard (lyophilized) 2 vials
    Stop Solution 1 x 8ml
    TMB One-Step Substrate Reagent 1 x 12ml
    TNF alpha Microplate (12 x 8 wells) 1 unit
  • Research areas

  • 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.
  • Involvement in disease

    Genetic variations in TNF are a cause of susceptibility psoriatic arthritis (PSORAS) [MIM:607507]. PSORAS is an inflammatory, seronegative arthritis associated with psoriasis. It is a heterogeneous disorder ranging from a mild, non-destructive disease to a severe, progressive, erosive arthropathy. Five types of psoriatic arthritis have been defined: asymmetrical oligoarthritis characterized by primary involvement of the small joints of the fingers or toes; asymmetrical arthritis which involves the joints of the extremities; symmetrical polyarthritis characterized by a rheumatoidlike pattern that can involve hands, wrists, ankles, and feet; arthritis mutilans, which is a rare but deforming and destructive condition; arthritis of the sacroiliac joints and spine (psoriatic spondylitis).
  • Sequence similarities

    Belongs to the tumor necrosis factor family.
  • Post-translational
    modifications

    The soluble form derives from the membrane form by proteolytic processing.
    The membrane form, but not the soluble form, is phosphorylated on serine residues. Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.
    O-glycosylated; glycans contain galactose, N-acetylgalactosamine and N-acetylneuraminic acid.
  • Cellular localization

    Secreted and Cell membrane.
  • Information by UniProt
  • Alternative names

    • APC1
    • APC1 protein
    • Cachectin
    • DIF
    • Differentiation inducing factor
    • Macrophage cytotoxic factor
    • Tnf
    • TNF superfamily member 2
    • TNF superfamily, member 2
    • TNF, macrophage derived
    • TNF, monocyte derived
    • TNF-a
    • TNF-alpha
    • TNFA
    • TNFA_HUMAN
    • TNFSF2
    • Tumor necrosis factor
    • Tumor necrosis factor (TNF superfamily member 2)
    • Tumor necrosis factor alpha
    • Tumor necrosis factor ligand superfamily member 2
    • Tumor Necrosis Factor, Membrane Form
    • Tumor necrosis factor, soluble form
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab100785 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Sandwich ELISA Use at an assay dependent concentration.

Images

  • Rat TNF alpha detected in cell lysates from NR8383.1 control cells (C) or cells stimulated for 6 hours with 5 ug x mL-1 of LPS (Sigma). Results shown for TNF alpha levels in 10e6 cells after background signal was subtracted (duplicates +/- SD).

  • Rat TNF alpha detected in cell supernatants from NR8383.1 control cells (C) or cells stimulated for 6 hours with 5 ug x mL-1 of LPS (Sigma); background signal subtracted (duplicates +/- SD).

  • Representative standard curve using ab100785

Protocols

References

This product has been referenced in:

  • Fahmi AA  et al. Chemical composition and protective role of Pulicaria undulata (L.) C.A. Mey. subsp. undulata against gastric ulcer induced by ethanol in rats. Heliyon 5:e01359 (2019). Read more (PubMed: 30957042) »
  • Kuwar R  et al. A novel small molecular NLRP3 inflammasome inhibitor alleviates neuroinflammatory response following traumatic brain injury. J Neuroinflammation 16:81 (2019). Read more (PubMed: 30975164) »
See all 21 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Yes, that lysis buffer will be good, the 150 mM sodium chloride, 1.0% Triton X-100, 50 mM Tris pH 8.0.


Here are some general guidelines.


Avoid using >0.1% SDS or other strongly denaturing detergents. In general, non-ionic detergents such as Triton X-100 or NP-40 are best, although zwitterionic detergents such as CHAPS, or mild ionic detergents such as sodium deoxycholate will work. 


Use no more than 2% v/v total detergent  


Avoid the use of sodium azide 


Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols 


We strongly recommend adding a protease inhibitor cocktail to the lysis buffer prior to homogenization.

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Question
Answer

Thank you for contacting us. The cell lysis buffer does not contain any protease inhibitor. Please let me know if you have any further questions.

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Answer



I can suggest the following general tissue lysate preparation procedure to prepare samples for both the kits.




Tissue lyste samples can be prepared using most conventional methods, for example with RIPA or other formulations.

Please note the following guidelines on lysis buffer composition:
1) Avoid using >0.1% SDS or other strongly denaturing detergents. In general, non-ionic
detergents such as Triton X-100 or NP-40 are best, although zwitterionic detergents such as
CHAPS, or mild ionic detergents such as sodium deoxycholate will work.
2) Use no more than 2% v/v total detergent
3) Avoid the use of sodium azide
4) Avoid using >10 mM reducing agents, such as dithiothreitol or mercaptoethanols

We strongly recommend adding a protease inhibitor cocktail to the lysis buffer prior to
homogenization. Most general biochemical supply companies including stock a wide variety of these products. Since susceptibility to proteolytic cleavage and the type of proteases present in the lysate vary, we do not recommend a specific product. Instead, your choice of which combination of protease inhibitors to use should be based upon a literature search for your protein(s) of interest and/or tissue or cell type. Phosphatase inhibitors may be used but are not necessary unless the antibodies used in the kit specifically
recognize phosphorylated forms of the protein.
Choices of the method for lysis and homogenization include glass-bead “smash,” douncing, freezethaw,
sonication and crushing frozen tissue with a mortar and pestle, or even a combination of these.
There is no best method for all sample types; your choice of method should be made following a brief
search of the literature to see how samples similar to yours have been prepared in previous
investigations.
After homogenization, centrifuge the lysates to remove cell/tissue debris (5 min @ 10,000 x g or 10
min @ 5,000 x g) and save the supernatant. Unless testing fresh, lysates should be frozen as soon as
possible and stored at -20°C (or -80°C, if possible). Centrifuge them again before incubating with any
immunoassay. Next, determine the protein concentration of your lysates using a total protein assay
not inhibited by detergents (such as the Bicinchoninic acid (BCA) assay) and normalize the volume of
each sample used to deliver the same amount of total protein for each assay.

Note: The Bradford assay is not recommended as it can be inhibited by the presence of detergents.
Since different cells and tissues may contain different amounts of protein, as starting point, we
suggest using 500 μL of lysis buffer per 1x106 cells or 10 mg tissue. You may have to adjust this
based upon your results. Your target total protein concentration of the homogenate should be at least
1,000 μg/mL, but 2,000 μg/mL or more would be better.

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Question
Answer

Thank you for your enquiry.

Please rest assured that there are no hazardous components in this kit according to Japanese law, and is safe for use by customers in Japan.

Have a nice day!

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Abreview for TNF alpha Rat ELISA Kit

Excellent Excellent 5/5 (Ease of Use)
Abreviews

Sample: Rat Tissue lysate - whole (spinal cord)
Specification: spinal cord
Amount of Sample: 100 µg
Type of Kit Used: ELISA
Positive control: Traumatized segment level T13
Did you use the Abcam Kit protocol?: Yes

Additional data
Additional Notes: I have used the kit with fresh rat spinal cord samples that underwent compression. The samples where homogenized a day before and kept in -20c. The strips allowed me to use the kit 3 times. The third time was a week after I have open Item F and still got a nice curve. R-square=0.9959.
Results are more than satisfying. It is a quick and straight forward assay.

Graph labels: Y axis is absorbance, X axis is pg/ml.The key labels are, from top to bottom: Standards, suppresses stds, standard curve.

Prof. Guy Sovak

Verified customer

Submitted May 21 2012

Question
Answer

Thank you for your enquiry.

The lysis buffer (item J) is used to prepare the cell and tissue sample. There is no mention on how to prepare your sample in the Protocol Booklet as most laboratories would have their own protocols and methods for doing so. We do recommend starting with 1mg/ml, preferable 2mg/ml volume (sample in lysis buffer) of sample. Please bear in mind that this is a starting point and the optimal concentration needs to be determined empirically.

You may also use your own lysis buffer. As a guideline, a lysis buffer must meet the following specifications.

A) has relatively low salt content (700 mM or less)
B) does not contain sodium azide
C) does not contain >0.1% SDS
D) does not contain >10 mM reducing agents (beta-mercaptoethanol or dithiothreitol)

I hope this information will be helpful.

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