Polyacrylamide Gel Electrophoresis-II
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During last lecture we studied about SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis) also called denaturing polyacrylamide gel electrophoresis. However, some times, we need to separate protein in non-denaturing conditions. This type of polyacrylamide gel electrophoresis is also called native gel electrophoresis because protein remains in native form even after electrophoresis. The basic difference in the native gel electrophoresis (native-PAGE) is the electrophoresis buffer does not contain SDS. Also, loading buffer does not have SDS and reducing agents and samples are not boiled. Moreover, discontinuous pH system is not used as protein focusing by isotachphoresis does not work well here (protein may have lower charge density than glycine).
We shall move ahead with few questions:
Question: What is the role of SDS in the SDS-PAGE?
Answer: It provides uniform negative charge to protein, so protein can migrate towards positive electrode during electrophoresis. In case of native-PAGE, proteins moves towards positive electrode due to its own charge.
Question: What is relationship between protein isoelectric point, pH and net charge on the protein? What is the pH of electrophoresis buffer?
Answer: pH of the electrophoresis buffer is 8.8. Any protein with isoelectric point above 8.8 will have negative charge and move toward positive electrode. They will be separated based on mass/charge ration. Proteins with isoelectric point below 8.8 may be separated by changing polarity of electrode. As protein focusing in stacking gel does not work well in native-PAGE, resolution of process is not very good (protein bands are not very sharp). However, the biggest advantage is that the proteins are separated in native form.
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