Bocillin-binding Assay for Membrane Proteins

Bocillin-binding assays on membrane extracts were performed as described previously [8 (link)]. For bocillin binding assays of purified proteins, 8.3 μM of protein and 250 μM Bocillin-FL (ThermoFisher cat# B13233) were incubated for 30 minutes at room temperature. The protein was then combined with sample buffer and 10 pmol/lane was run on a 4–20% polyacrylamide gel. Bocillin gels were imaged using a Typhoon 9500 fluorescence imager (GE Healthcare) with excitation at 488 nm and emission at 530 nm.

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Rohs P.D., Buss J., Sim S.I., Squyres G.R., Srisuknimit V., Smith M., Cho H., Sjodt M., Kruse A.C., Garner E.C., Walker S., Kahne D.E, & Bernhardt T.G. (2018). A central role for PBP2 in the activation of peptidoglycan polymerization by the bacterial cell elongation machinery. PLoS Genetics, 14(10), e1007726.

Publication 2018

Bocillin-FL Typhoon 9500 fluorescence imager

Assays Binding proteins Bocillin fl Buffer Fluorescence Gels Membrane Polyacrylamide gel Protein Typhoon

Corresponding Organization : Howard Hughes Medical Institute

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Variable analysis

independent variables

Concentration of Bocillin-FL (250 μM)

dependent variables

Bocillin binding to purified proteins

control variables

Concentration of purified protein (8.3 μM)
Incubation time (30 minutes)
Temperature (room temperature)
Gel electrophoresis (4-20% polyacrylamide gel, 10 pmol/lane)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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