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    tnf-alpha-affibody-molecule-agarose-ab31909.pdf

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Immunology Innate Immunity Cytokines TNF Superfamily
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Anti-TNF alpha Affibody® Molecule (Agarose) (ab31909)

  • Datasheet
Q&A (1)References (1)

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Immunoprecipitation - Anti-TNF alpha Affibody® Molecule (Agarose) (ab31909)

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    Overview

    • Product name

      Anti-TNF alpha Affibody® Molecule (Agarose)
      See all TNF alpha affibody® molecule
    • Conjugation

      Agarose
    • Conjugation notes

      This molecule is immobilized on agarose at the unique C-terminal cysteine.
    • Tested applications

      Suitable for: IPmore details
    • Species reactivity

      Reacts with: Human
    • Immunogen

      Recombinant full length protein corresponding to Human TNF alpha. Recombinant protein produced in E. coli.

    • General notes

      ab31909 is a recombinant protein produced in E. coli.

      What are Affibody Molecules?
      Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants. Thus, the libraries consist of a multitude of protein ligands with an identical backbone and variable surface- binding properties. The current Affibody® libraries contains billions of variants. In function, Affibody® molecules mimic antibodies, nature’s own binders to an infinite number of antigens. Compared to antibodies, the most striking dissimilarity of Affibody® molecules is the small size. Affibody® molecules have a molecular weight of 14 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of Affibody® molecules is similar to that of an antibody. The advantages of Affibody® molecules over antibodies are · their small size · the simple structure of the molecules · its robust physical properties · its ability to fold correctly intracellularly · the fast and cost-efficient production in bacteria · the possibility to produce Affibody® molecules through chemical synthesis · the possibility to couple Affibody® molecules in multimeric constructs

    Properties

    • Form

      Liquid
    • Storage instructions

      Shipped at 4°C. Store at +4°C. Do Not Freeze.
    • Storage buffer

      pH: 7.50
      Preservative: 0.02% Sodium azide
      Constituents: 0.328% Sodium phosphate, 0.87% Sodium chloride
    • Concentration information loading...
    • Purification notes

      ab31909 is >98% as determined by SDS-PAGE and RP-HPLC analysis.
    • Affibody® molecule notes

      What are Affibody Molecules?
      Affibody® affinity ligands are small, simple proteins composed of a three-helix bundle based on the scaffold of one of the IgG-binding domains of Protein A. Protein A is a surface protein from the bacterium Staphylococcus aureus. This scaffold has excellent features as an affinity ligand and can be designed to bind with high affinity to any given target protein. The domain consists of 58 amino acids, 13 of which are randomized to generate Affibody® libraries with a large number of ligand variants. Thus, the libraries consist of a multitude of protein ligands with an identical backbone and variable surface- binding properties. The current Affibody® libraries contains billions of variants. In function, Affibody® molecules mimic antibodies, nature’s own binders to an infinite number of antigens. Compared to antibodies, the most striking dissimilarity of Affibody® molecules is the small size. Affibody® molecules have a molecular weight of 14 kDa, compared to the molecular weight of antibodies, which is 150 kDa. In spite of its small size, the binding site of Affibody® molecules is similar to that of an antibody. The advantages of Affibody® molecules over antibodies are · their small size · the simple structure of the molecules · its robust physical properties · its ability to fold correctly intracellularly · the fast and cost-efficient production in bacteria · the possibility to produce Affibody® molecules through chemical synthesis · the possibility to couple Affibody® molecules in multimeric constructs
    • Research areas

      • Immunology
      • Innate Immunity
      • Cytokines
      • TNF Superfamily
      • Signal Transduction
      • Growth Factors/Hormones
      • TNF
      • Microbiology
      • Organism
      • Virus
      • RNA Virus
      • ssRNA positive strand virus
      • SARS Coronavirus
      • Cancer
      • Growth factors
      • TNF
      • Cardiovascular
      • Atherosclerosis
      • Vascular Inflammation
      • Inflammatory mediators
      • Metabolism
      • Types of disease
      • Metabolic disorders
      • Neuroscience
      • Development
      • Neuroscience
      • Diseases
      • Neuroscience
      • Processes
    • 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.
    • Target information above from: UniProt accession P01375 The UniProt Consortium
      The Universal Protein Resource (UniProt) in 2010
      Nucleic Acids Res. 38:D142-D148 (2010) .

      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

      • Entrez Gene: 7124 Human
      • Omim: 191160 Human
      • SwissProt: P01375 Human
      • Unigene: 241570 Human

      Associated products

      • Recombinant Protein

        • Recombinant human TNF alpha protein (Active) (ab259410)
        • Recombinant mouse TNF alpha protein (Active) (ab259411)
      • Related Products

        • Human TNF alpha ELISA Kit (ab181421)
        • Human TNF alpha Matched Antibody Pair Kit (ab213467)

      Applications

      The Abpromise guarantee

      Our Abpromise guarantee covers the use of ab31909 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
      IP
      Use at an assay dependent concentration.

      The molecule is immobilized on agarose at the unique C-terminal cysteine.

      This agarose immobilized Anti-TNF alpha Affibody® molecule is excellent for immunoprecipitation studies of TNF alpha in cell extracts or other solutions that contain TNF alpha proteins. Can also be used for affinity chromatography.

      Notes
      IP
      Use at an assay dependent concentration.

      The molecule is immobilized on agarose at the unique C-terminal cysteine.

      This agarose immobilized Anti-TNF alpha Affibody® molecule is excellent for immunoprecipitation studies of TNF alpha in cell extracts or other solutions that contain TNF alpha proteins. Can also be used for affinity chromatography.

      Images

      • Immunoprecipitation - Anti-TNF alpha Affibody® Molecule (Agarose) (ab31909)
        Immunoprecipitation - Anti-TNF alpha Affibody® Molecule (Agarose) (ab31909)

        The agarose immobilized Anti-TNF alpha Affibody® molecule precipitates TNF-alpha protein from cell extracts derived from LPS stimulated but not from unstimulated THP-1 cells.

        RESULTS

        IMMUNOPRECIPITATION

        The human pro-monocytic cell line THP-1 was primed with PMA (200 nM) over night and stimulated with LPS (200 ng/ml) or with PBS (control) for 2 hours prior to termination of culture. Cell extracts prepared from LPS stimulated cultures and from control cells were incubated with agarose immobilized Anti-TNF-alpha Affibody® molecule for 2 hours. After incubation, the unbound proteins were washed away and the bound protein was eluted with SDS-PAGE separation and blotted onto a PVDF filter. The filter was stained with an antibody against full length TNF-alpha protein (25 kD).

        Figure 1a and 1b show that TNF-alpha was precipitated from LPS stimulated, but not from PBS stimulated THP-1 cell extract, using agarose immobilized Anti-TNF alpha Affibody® molecule. With increased amount of total protein, the TNF-alpha band became more intense. TNF-alpha was not detected in cell extracts prior to immunoprecipitation proving that an immunoprecipitation step is needed for TNF-alpha detection in this model system.

        These results show that the Anti-TNF alpha Affibody® molecule efficiently precipitates TNF-alpha proteins from a complex mix of proteins. When performing immunoprecipitation experiments with antibodies, there is often a problem with cross reactions between the enzyme conjugated second step reagent and the precipitating antibody. This type of cross reaction is avoided with an Affibody® molecule as the precipitating reagent.

      Datasheets and documents

      • Datasheet download

        Download

      Protocols

      • TNF alpha Immunoprecipitation

      Click here to view the general protocols

      References (1)

      Publishing research using ab31909? Please let us know so that we can cite the reference in this datasheet.

      ab31909 has been referenced in 1 publication.

      • Jiang H  et al. SIRT6 regulates TNF-a secretion through hydrolysis of long-chain fatty acyl lysine. Nature 496:110-3 (2013). PubMed: 23552949

      Customer reviews and Q&As

      Q&A
      Submit a question

      Question

      Can you tell if this product contains cysteine blocked agarose?

      Read More

      Abcam community

      Verified customer

      Asked on Sep 14 2007

      Answer

      Thank you for your enquiry. I am sorry but we do not know this product contains cysteine blocked agarose. However, I can confirm that for immobilization of the Anti-TNF-alpha Affibody® Molecule on an agarose matrix we employ Pierce's SulfoLink Coupling Gel (www.piercenet.com) and after the coupling reaction the agarose is routinely blocked with the supplied blocking buffer.

      Read More

      Abcam Scientific Support

      Answered on Sep 14 2007

      Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
      For licensing inquiries, please contact partnerships@abcam.com

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