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Other Immunogen Type corresponding to Human TNF alpha. 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
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.
|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.
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.
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.