Native Isolation of 3×HA‐Tagged Protein Complexes to Characterize Protein‐Protein Interactions

Improved native isolation of endogenous Protein A-tagged protein complexes.

Here we report a modified peptide reagent useful for the rapid, native elution of protein complexes containing a Protein-A-tagged component. We tested this reagent for the elution of tagged endogenous protein complexes from yeast (Nup53p/Nup170p dimer; Nup1p/Kap95p/Kap60p trimer; pentameric GINS complex) and bacteria (RNAP holoenzyme). The majority of the affinity-isolated material is released ...

Engineered high-affinity nanobodies recognizing staphylococcal Protein A and suitable for native isolation of protein complexes.

In addition to its high affinity for antibody Fc domains, staphylococcal Protein A has been shown to bind certain Fab domains. We investigated this in order to develop a small, recombinant Protein A-binding alternative to immunoglobulin G (IgG) from nanobodies, single-domain antibodies derived from a camelid variant IgG's variable region. We engineered a nanobody with affinity solely for Protei...

Design of Oligosaccharide Libraries to Characterize Heparan Sulfate – Protein Interactions

Native electrophoretic techniques to identify protein-protein interactions.

Permanent protein-protein interactions are commonly identified by co-purification of two or more protein components using techniques like co-immunoprecipitation, tandem affinity purification and native electrophoresis. Here we focus on blue-native electrophoresis, clear-native electrophoresis, high-resolution clear-native electrophoresis and associated techniques to identify stable membrane pro...

Native fractionation: isolation of native membrane-bound protein complexes from porcine rod outer segments using isopycnic density gradient centrifugation.

Networks of interacting protein control physiological processes in all living cells. Considerable effort has recently been invested in understanding protein interactions under normal and diseased conditions. One approach to elucidate the composition of protein complexes is native fractionation followed by immunological or MS-based identification of individual compounds. Native fractionation, in...