Hello everyone,
Has someone experienced before, unspecific electrostatic interactions of a protein with high pKa (8.8) with E.coli cell membrane? I am using bacterial display of E.coli for peptide display. When I try to find a binding partner of the eGFP-conjugated enzyme to the displayed peptide, I see a unspecific binding of the protein the cell membrane of E.coli. I tried to disrupt the interactions using salt (1M NaCl, 200 mM MgCl2) and higher pH 8.5 HEPES buffer. Eventhough most of the interactions are gone, but in FACS I still can see unspecific binding on single cell level. Any body has a better idea?
Dear
Some relatives paper concerning your question:
-Structural Basis of Perturbed pKa Values of CatalyticGroups in Enzyme Active Sites
Thomas K. Harris and George J. Turner. IUBMB Life, 53: 85–98, 2002
http://www.medschool.lsuhsc.edu/biochemistry/Courses/Biochemistry201/Haas%20Files/Harris5674.pdf
http://biochemistry2.ucsf.edu/programs/ptf/prologue%20links/Diff%20&%20Trans%20Membranes.pdf
using a mild detergent in your binding buffer (0.1% triton or tween) might allow you to see only the specific interactions.
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