Lab 2 - Protein Isolation Flashcards
In order to sudy a particular protein, it must be _
isolated in a relatively pure state
one method to isolate a particular protein from a mixture of soluble substances is through _
protein precipitation
this method capitalizes on the solubility of proteins in a solution
protein precipitation
two commonly used techniques of protein precipitation
salting-out method
isoelectric precipitation
In salting out process, proteins are usually precipitated out of the solution by saturating the protein solution with _ or _
ammonium sulfate
magnesium sulfate
True or False:
By increasing the ionic strength of the solution, the solubility of the protein increases.
False
solubility decreases
True or False: When the ionic strength of the solution increases, water molecules tend to interact more with the ions from the added salt, and protein molecules are left to interact with one another.
True
Variation in pH changes the ionization state of the _ in a protein, which also changes its net charge
functional groups
Proteins are least soluble in a solution _
equal to its isoelectric pH
proteins are soluble in pH _
greater than or less than the isoelectric point
characterized by the net charge of zero
isoelectric point (IpH)
protein found in egg whites
Ovalbumin
how would you precipitate ovalbumin?
using ammonium sulfate precipitation
How will you precipitate casein?
using isoelectric precipitation
What is the purpose of the following reagents/steps in the salting-out technique?
Filtering through cheesecloth
removes large particles from the egg white to ensure clearer filtrate before precipitation
What is the purpose of the following reagents/steps in the salting-out technique?
1M acetic acid
adjust the pH and denature some proteins allowing for better precipitation by reducing solubility
What is the purpose of the following reagents/steps in the salting-out technique?
Ammonium sulfate
ammonium sulfate is a salt used to increase ionic strength of the solution, reducing solubility that cause them to precipitate
What is the purpose of the following reagents/steps in the salting-out technique?
Ice bath
Ice bath helps slow down molecular motion, stabilizing proteins and preventing denaturation during precipitation.
enhances formation of aggregates for easier collection
Explain the effect of increasing ammonium sulfate on the solubility of ovalbumin. What is the purpose of bringing the solution to 40% to 60% saturation?
as ammonium sulfate concentration increases, ionic strength also increases, reducing solubility of ovalbumin, causing it to precipitate.
the solution is brought to 40% to 60% saturation because different proteins have varying solubility, and this range ensures selective precipitation of ovalbumin without unwanted contaminants.
Discuss the purpose of the following in isoelectric precipitation technique
Centrifugation at 4000 rpm
separates solid and liquid components, allowing for isolation of proteins that have precipitated out of solution.
The highspeed rotation forces denser particles (precipitated proteins) to settle at the bottom.
Discuss the purpose of the following in isoelectric precipitation technique
0.5 NHCl
HCl is added to lower the pH of the solution to 4.8, which is the isoelectric point of casein. At this pH, casein has no charge and is insoluble causing it to precipitate
Discuss the purpose of the following in isoelectric precipitation technique
washing with diethyl ether and ethanol
removes impurities to ensure that isolated casein is pure and free from contaminants
What is isoelectric pH? How can we use the IEP in isolating a particular protein
Isoelectric pH is the pH where proteins has no net charge, making it least soluble in water. by adjusting pH of a solution to the protein’s IpH, we can actively precipitate the protein to isolate.
advantages of salting out precipitation
simple and cost-effective
used for large-scale protein isolation
allow selective precipitation based on solubility differences
disadvantages of salting-out precipitation
requires control of salt conc.
cause protein denaturation if not properly handled.
additional purification steps may be necessary to remove excess salt
advantages of isoelectric precipitation
highly selective
isolate proteins with minimal contamination
does not require high salt conc, reducing the need for desalting steps
disadvantages of isoelectric precipitation
requires precise pH control
some proteins may aggregate irreversibly
may not be suitable for all proteins
