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Experiments with increasing signals are SSB proteins binding to fluorescently labeled DNA substrate. Experiments with decreasing signal are intrinsic trp changes in the SSB protein upon binding to DNA.
Impressively, the students setup three different conditions within each well. Long and arduous experiment....hoping we get some crystal hits!!
Students tested whether their SSB proteins wrapped DNA and if they have the two DNA binding modes similar to E.coli SSB. Experiments were done using a stop flow instrument where 1 uM SSB was rapidly mixed with 20 nM DNA containing a Cy3-Cy5 FRET pair. Wrapping = high FRET, formation of the differential binding modes is determined if there is a loss in the FRET signal. One SSB protein from Bacillus anthracis does not the two binding modes!! Students monitored binding of their SSB protein to a Cy3-labeled DNA substrate. The Cy3 fluorescence changes upon binding to protein and the change in fluorescence is measured as a function of time in a Applied Photophysics SX20 Stop flow instrument. Excitation is 530 nm and emission is collected using a 570 nm long pass filter. The DNA concentration is held constant at 500 nM and the SSB protein concentration varied from 0.05 uM to 2 uM SSB. All concentrations are pre-mixing. |
AuthorDr. Edwin Antony and Dr. Sofia Origanti has put together this course. The material presented here are generated by the students of the BIOL 4102 class at Marquette University. (Fall 2015). Improvements can only be made if there is adequate feedback and we thank you in advance for your time. Archives
November 2016
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