Tutorial 6-Protein Purification Flashcards
Discuss the chemical basis of the ion-exchange chromatography?
Proteins bind to an anion exchanger (positively charged) at pH values above their isoelectric point (pI) and to a cation exchanger (negatively charged) at pH values below the pI.
Separates protein based on their charges on a particular pH.
Depends on the ionized side chains.
Why are the scientist still use the traditional purification methods over many sophisticated purification methods?
Traditional purification procedures make use of the physiochemical properties of the enzyme of interest for elucidating enzyme mechanism and solving protein three dimensional structures and for preparation of highly pure biocatalyst.
- Enables determination of the amount of given enzyme in units
- Specific activity is a good indication of the purity and quality of the enzyme preparation.
- The purification factor provides an insight into the efficiency of each step.
- It is inexpensive
What are the properties of proteins that we could use them as separation methods for the proteins?
- Charge
- Molecular size
- Bio-affinity
- Absorption principles
- Selective precipitation
Differentiate the novel protein purification methods over the traditional purification methods?
Novel protein purification methods have better separation efficiency and avoids problem of mobile phase compatibility compared to traditional purification methods.
Cost is reduced due to re-usability in modern techniques but modification must be performed. For example, to use MNP stably in protein purification, surface modification must be performed.
Compared with traditional cell separation methods and column chromatography methods, purification can be performed without cell separation, saving resources, operation time and equipment.
Modern techniques involve a variety of chips based on electrophoretic or chromatographic principle separation, while most traditional procedures start with ammonium sulfate fractionation.
Traditional, due to handling, protein can change and protein functionality can be lost. Modern methods involve less handling and result in less errors.
What is the main principle, that is considered in the salting out method using the ammonium sulphate.
Concentration
Solubility
When high concentrations of small, highly charged ions such as ammonium sulfate are added, these groups compete with the proteins to bind to the water molecules. It removes the water molecules from the protein and decrease its solubility, resulting in precipitation.
Depending on the protein, different amount of salt concentration is required for salting out.
For example, 0.8 M ammonium sulfate precipitates fibrinogen, whereas 2.4 M is needed to precipitate serum albumin.
What are the reasons for using the column chromatography widely for the industrial purposes even though there are many novel techniques are introduced?
In industries, the use of column chromatography method was crucial for purification due to its large resolving power and high recovery of enzyme activity. Isolate active ingrediant, impurity is removed, process can be completely automated, stationary phase is inexpensive and can be reused.
What are the common fractionation methods used for the isolation of the protein of interest?
- Salting Out
- Dialysis
- Gel-filtration chromatography
- Affinity chromatography
- High-performance liquid chromatography
- Electrophoresis.
Briefly explain the steps of purification of a crude protein sample to the pure protein sample using any selected purification method?
Due to it being crude protein, purification must take place, fractionation method can be used.
Differential solubilization method with the ammonium sulfate.
Salting out method
Ultrapurification
Ultracentrifugation, high speed, unwanted materials are removed from crude sample.
Then chromatography method can be used.
What are the facts that we expect during successful purification scheme
Good affinity and purity
Sensitivity maintained, precision, substrate availability, active resin
Cost
Stability maintained
A purified protein sample contains 10 μg of protein and has an enzyme activity of 1 m mole of ATP synthesized/sec (1 unit). What is the specific activity of the final purified sample
ug to m (unit)
10 x 10^-3 =
Divide activity(1m) by 10 ug (change units).
What are the best methods of purification of the [proteins with large differences in molecular
weight?
Affinity chromatography
Size-exclusion chromatography
Mention the purification methods that are widely used the separation based on the net charge?
Ion-exchange chromatography
there is second one
What are the common detergents used to disrupt the cell membranes of the cells during the
protein purification
- SDS
- ethyl trimethyl ammonium bromide
What are the properties of the proteins that are compatible with the gel filtration technology for
the purification?
Molecular size
Molecular weight
