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What are Affibody Molecules?
Affibody® affinity ligands are unique research reagents, produced using innovative protein-engineering technologies. They 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. In function, Affibody® Molecules mimic monoclonal antibodies. Compared to antibodies, the most striking dissimilarity of Affibody® Molecules is the small size. Affibody® Molecules have a molecular weight of 14kDa, compared to the molecular weight of antibodies, which is 150kDa. In spite of its small size, the binding site of Affibody® Molecules is similar to that of an antibody. The advantages of Affibody® Molcules over antibodies are: -their small size -the simple structure of the molecules -its robust physical properties; able to withstand a broad range of analytical conditions, including extreme pH and elevated temperature -its ability to fold correctly intracellularly -the fast and cost effective production in bacteria -the potential to couple Affibody® Molecules in multimeric constructs Affibody® Molecules have highly competitive properties for applications within affinity purification, sample preparation, protein detection and in vitro diagnostics.
Our Abpromise guarantee covers the use of ab31892 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 dilution.
AFFIBODY® MOLECULES ARE PROTOCOL SPECIFIC. PLEASE REFER TO THE "PROTOCOLS" SECTION.
The agarose immobilized Anti-ErbB2 Affibody® molecule precipitates ErbB2-protein from cell extracts derived from ErbB2 positive cell lines SK-BR-3 and HEP-G2, but not from the ErbB2 low expressing SH-SY5Y cell line.
Cell extracts prepared from SK-BR3, HEP-G2 and SH-SY5Y cells were incubated with agarose immobilized Anti-ErbB2 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 the intracellular domain of the ErbB2 receptor. The antibody binds to both the fulllength protein (150 kDa) and to a splice variant (approximately 100 kDa) of the receptor that has been reported to be produced by several cell lines.
Figure 1a shows that the ErbB2 proteins were precipitated from different volumes of SK-BR3 cell extract using the agarose immobilized Anti-ErbB2 Affibody® molecule. An intense ErbB2 band was obtained on the Western blot from as little as 25 μl extract. With increased volume of extract, the ErbB2 band became even more intense. ErbB2 proteins were also precipitated from HEP-G2 cell extract, show in figure 1b. A relatively strong band was obtained when precipitating from 300 μl extract indicating that HEP-G2 expresses ErbB2 to a lower level than SK-BR3. No bands were visible after precipitation with SH-SY5Y cell extracts, shown in figure 1 c, suggesting that this cell line has a very low level of ErbB2 expression.
These results show that the Anti-ErbB2 Affibody® molecule efficiently precipitates ErbB2 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.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"