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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 ab36039 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. This molecule can be used for immunoprecipitation of EGFR from complex mixes of proteins.|
|ICC/IF||Use at an assay dependent dilution.|
|Flow Cyt||Use at an assay dependent dilution.|
Cell extracts were prepared from high EGFR expressing human squamos carcinoma cell line A431 , from low EFGR expressing SH-SY5Y (human neuroblastoma) cells and from the EGFR negative RAMOS (human B-cell lymphoma) cell line. The three extracts were incubated with agarose immobilized Anti-EGFR Affibody® molecule for 2 hours. After incubation, the unbound proteins were washed away and the bound protein was eluted and separated with SDS-PAGE and blotted onto a PVDF filter. The filter was stained with an antibody against full length EGFR, approximately 1 40 kDa.
A single protein with MW of approximately 40 kDa was precipitated from A431 and SH-SY5Y cells but not from RAMOS cells, as shown in figure . With increased amount of A431 proteins, the 40 kDa EGFR band got more intense on the Western blot (lanes -3). The EGFR band was also detected in A431 cell extracts prior to precipitation together with bands of lower MW that were not present after precipitation (lane 4). SH-SY5Y cells also express EGFR but to a lower level than A431 , as shown by a relatively weak EGFR band (lane 5). There were no EGFR detected in SH-SY5Y cell extracts prior to precipitation (lane 6) and there were no EFGR precipitated from RAMOS cells or present in the cell extracts (lanes 7-8).
In summary, the Anti-EGFR Affibody® molecule efficiently precipitates EGFR from a complex protein mix. When performing immunoprecipitation experiments with antibodies, there is often a problem with cross reaction between the enzyme conjugated second step reagent and the precipitating antibody but this type of cross reaction is elegantly avoided using an Affibody® molecule as the precipitating reagent.
ab36039 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"