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ESCORTing proteins directly from whole cell-lysate for single-molecule studies.

Anal Biochem.. 2017-10; 
Srinivasan S, Hazra JP, Singaraju GS, Deb D, Rakshit S.
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Catalog Antibody ... (iii). Introducing polyglycine on the surface to harness proteins via the sortase mechanism. The coverslips and cantilevers were incubated in 100 μM tetraglycine to N-terminus along with cysteine at C-terminus (GGGGC) (GenScript). ... Get A Quote

摘要

We have developed a method for Enzymatic Sortase-assisted Covalent Orientation-specific Restraint Tethering (ESCORT) recombinant proteins onto surfaces directly from cell-lysate. With an improved surface passivation method, we obviate the cumbersome purification steps even for single molecule studies that demand high purity in the sample. We demonstrated high-specificity of the method, high-passivity of the surface and uncompromised functional integrity of anchored proteins using single molecule fluorescence and force-mapping. We anticipate that this method will substantially reduce the investment by way of time, money and energy in the area of single molecule studies.

关键词

Protein-protein interactions; Pull-down assays; Surface modification