Hi, I am learning to perform footprinting experiment. In the protocol I have there are the following components of the reaction buffer: HEPES buffer (7.8 pH), glycerol, BSA, DTT, KCl, MgCl2, CaCl2. Could you please help me understanding the role of these components?
Based on my knowledge,
- DTT should act as reducing agent that prevents the oxidation of thiol groups on cysteine residues in proteins (such as DNase I), helping them maintaining their active, reduced state.
- BSA should prevent nonspecific adsorption of enzymes to the walls of reaction tubes and surfaces, helping them to maintain their activity.
- KCl: I don't know xD
- CaCl2 and MgCl2 provide cofactor for DNase I. Moreover, magnesium ions help stabilize the DNA structure.
- Glycerol should help to maintain enzyme activity at lower temperatures and can also influence the viscosity of the solution, which might affect the mobility of DNA and enzymes during the reaction.
Is it correct? Are there any other roles played by these components? Is any of them less important in the buffer?
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