quiz Q pool #5 Flashcards
What is the charge on an acidic amino acid at pH 7
neg
What is the charge on a basic amino acid at pH 7
pos
What is the charge on a protein when:
a. the pH is equal to the protein’s pI?
b. the pH is above the protein’s pI?
c. the pH is below the protein’s pI?
a. no net charge
b. neg
c. pos
If you have a negatively charged ion-exchange column such as CM-cellulose and you are using it to isolate a particular protein, would you like the pH to be above or below that protein’s pI? WHY
Below, because if the pH is below than the charge on the proteins is positive and will bind the the negatively charged column strongly.
) What are 2 ways to elute proteins from an ion-exchange column? Briefly, how do they work?
- pH gradient: control the charge on the proteins, thus affecting affinities for the ion exchanger due to differences in their net charge
- salt gradient: depending on ionic strength, the competition between buffer ions and the proteins for charged groups on the ion-exchanger is decreased or increased
How do you determine a protein’s molecular weight using SDS-PAGE? Be specific as to the steps involved! Hint: the first step is running an SDS-PAGE gel with the protein whose MW is to be determined in one lane and a series of MW standards in another lane. List the SUBSEQUENT steps.
The first step is running an SDS-PAGE gel with the protein whose MW is to be determined in one lane and a series of MW standards in another lane
• Using the discontinuous system, the proteins stack on top of one another, and are sorted into respective stacks. Next, the proteins travel based on size/MW.
• One then measures the relative mobility using Rf= Distance of protein migration/distance of traveling dye migration.
• Then we plot this on semi-log paper to generate a calibration curve against which MW of known protein can be determined.
To generate a standard curve with your molecular weight standards, plot ? on the log scale vs. ? on the linear scale of your semi-log paper. (2 points)
MW and Rf (relative mobility)
What are the 2 steps in two-dimensional (2D) gel electrophoresis and on what basis are proteins separated in each?
gel electrophoresis is separation via isoelectric focusing (IEF). IEF
separates proteins based on their pI (isoionic point). The proteins
move through a pH gradient in an electric field and stop where the pH
of the surrounding buffer equals that of the protein. The second part
is SDS-PAGE which separates proteins by size
Early in purification, two low resolving steps often used are:
The early steps in purification scheme are usually those which have the lowest resolving power such as ammonium sulfate precipitation, or for some proteins, heat denaturation
Traditionally, one unit of enzyme activity is defined as:
that which causes the conversion of 1 micromol of substrate per minute under the (optimal) conditions of measurement.
The number of units of enzyme activity per milligram of total protein is the ______of the enzyme
specific activity
Enzyme yield is defined as:
Yield = units of enzyme after purification step/
units of enzyme in the original preparation x 100
We will consider our purification to be complete when the apparently pure protein
yields one spot when tested by
2D Gel Electrophoresis
) DEAE cellulose or CM cellulose are only effective in the pH range of _ to _. The starting buffer should be of reasonably ___ionic strength, because the affinity of proteins for the ion exchange resins INCREASES or DECREASES as ionic strength increases
5 to 9
low
decreases
When choosing a gel type for optimal fractionation of complex proteins the gel pore size is such that the desired protein is
ispartially excluded from the gel beds, a condition which leads to the greatest degree of fractionation from other protein species.
