Answer
a) Specific Activity = amount of protein of interest (pmol or enzyme units) / amount of total protein % Yield = amount of protein of interest obtained /original protein present in the crude extract Fold Purification = specific activity of purified protein / crude amount.
b) DEAE is a weak anion exchanger. This exchange is used to separate proteins that have faintly differing charges. The resin carries positive charge and hence interacts favourably with negative charges.Positive charge of the resin is due to the protonated amine group. .This kind of electrostatic interaction is dependent on the pH range and should be less than the pka of amine group. Also such a negative charge should also be found in the protein to be extracted from a crude preparation. Hence using DEAE would cause greater loss of Mb compared to using the affinity chromatography method because affinity chromatography is based on the specific binding of the protein to the stationary phase and hence the product will be eluted out last.
c) Affinity Chromatography.
d) Affinity Chromatography.
Work Step by Step
a) Specific Activity = amount of protein of interest (pmol or enzyme units) / amount of total protein % Yield = amount of protein of interest obtained /original protein present in the crude extract Fold Purification = specific activity of purified protein / crude amount.
b) DEAE is a weak anion exchanger. This exchange is used to separate proteins that have faintly differing charges. The resin carries positive charge and hence interacts favourably with negative charges.Positive charge of the resin is due to the protonated amine group. .This kind of electrostatic interaction is dependent on the pH range and should be less than the pka of amine group. Also such a negative charge should also be found in the protein to be extracted from a crude preparation. Hence using DEAE would cause greater loss of Mb compared to using the affinity chromatography method because affinity chromatography is based on the specific binding of the protein to the stationary phase and hence the product will be eluted out last.
c) Affinity Chromatography.
d) Affinity Chromatography.
