Home assignment: A survey on activity staining of few enzymes, like proteases, lipases etc |
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-Native-PAGE is very useful for subunit mass/stoichiometry determination of oligomeric proteins. As in native-PAGE generally oligomeric proteins retains their tertiary structure, we get oligomeric mass. In SDS-PAGE subunits are dissociated when boiled with SDS and reducing agent. Thus, SDS-PAGE gives monomeric mass. A protein shows a band corresponding to 100kDa in native-PAGE but 25kDa in SDS-PAGE suggest the protein is an oligomeric protein with four identical subunits.
Question: A protein gives 100 kDa molecular mass in native-PAGE but two bands in SDS-PAGE corresponding to 25 kDa and 50 kDa. How many subunits are there in oligomeric protein. Write molecular mass of each subunits
Answer: Total three subunits. Two subunits of 25 kDa and one of 50 kDa
Non-reducing SDS-PAGE: This is a modified version of SDS-PAGE where reducing agents are not used. This provides some information about disulfide bond pattern in oligomeric proteins. Let us try to understand the application by a simple question.
A 100 kDa protein is trimer composed of two molecule of 25 kDa and single poly peptide of 50 kDa. A non reduced SDS-PAGE gives a band of 100 kDa (equivalent to native-PAGE). However, reduced SDS-PAGE gives two bands one equivalent to 25 kDa and other 50 kDa .
What information do you get from the experiment?
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