Homework Answers
We can compare mobility of purified protein in SDS under reducing and non reducing conditions. The protein having disulfide bond will exhibit higher mobility in non reducing condition because the presence of disulfide bonds will make them more compact.
Experiment:A
1.start by blocking free thiols in given protein samples with N-ethylmaleimide and remove the excess.
2. Denature the protein with urea or SDS.
3.devide denature protein into 2 parts.
4 leave one part oxidized and reduce the other part with TCEP.
5 React both parts with fluorescein maleimide.
6. Run the samples with SDS-PAGE.
Observation:
If there may be a disulfide bond in the protein, the TCEP-reduced part will give fluorescent and the oxidized part will not give.
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