1.) Explain the purpose of each of the following components of the lysis buffer: Lysozyme, NP-40, DNAse I, Mg2+ and Ca2+.
2.) What role does salt play during lysis ? what would happen if the 0.3 M NaCl were omitted ?
3.) How would the quality of purification change if the HisTrap column were charged with zinc or cobalt instead of nickel?
4.) When observing the final purified protein on a gel, why do you run a range of protein masses ?
5.) Why must protein be denatured with SDS to determine its molecular weight by using electrophoresis ?
6.) What is the pI of a protein ? Can the pI be used to purify a protein ? Explain.
7.) High salt concentration (eg. 3.6 M ammonium sulfate) is often used to precipitate protein for long-term storage. From this state the protein can be re-dissolved and exchanged into an appropriate buffer. How does the high salt concentration result in protein precipitation?
8.) Protective reagents are often added to samples of protein during purification and for storage. Explain why each of the following reagents may be used for storing proteins : B-ME or DTT, EDTA, glycerol, and DMSO/
9.) Proteins typically exhibit absorption peaks at 215 nm and 280 nm. What gives rise to these peaks?
1.) Explain the purpose of each of the following components of the lysis buffer: Lysozyme, NP-40, DNAse I, Mg2+ and Ca2+.
2.) What role does salt play during lysis ? what would happen if the 0.3 M NaCl were omitted ?
3.) How would the quality of purification change if the HisTrap column were charged with zinc or cobalt instead of nickel?
4.) When observing the final purified protein on a gel, why do you run a range of protein masses ?
5.) Why must protein be denatured with SDS to determine its molecular weight by using electrophoresis ?
6.) What is the pI of a protein ? Can the pI be used to purify a protein ? Explain.
7.) High salt concentration (eg. 3.6 M ammonium sulfate) is often used to precipitate protein for long-term storage. From this state the protein can be re-dissolved and exchanged into an appropriate buffer. How does the high salt concentration result in protein precipitation?
8.) Protective reagents are often added to samples of protein during purification and for storage. Explain why each of the following reagents may be used for storing proteins : B-ME or DTT, EDTA, glycerol, and DMSO/
9.) Proteins typically exhibit absorption peaks at 215 nm and 280 nm. What gives rise to these peaks?
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