Exam 2 Flashcards
the ratio of enzyme activity relative to total protein
specific activity
a type of purification that is based on the attraction of the protein for a particular chemical gorup
affinity chromatography
proteins can be separated from small molecules and ions through a semipermeable membrane by
dialysis
in gel filtration chromatography, in what order to proteins come off of the column
large -> medium -> small
allows the detection of small amounts of target proteins as well as the ability to determine the size of target proteins
western blotting
what is the gel matrix for protein electrophoresis
polyacrylamide
the pH of a protein at which its net charge is zero
pI or isoelectric point
an immunological technique for sensitive quantification of a target protein
ELISA
What is the advantage of adding SDS to gel electrophoresis
SDS allows proteins to be separated on the basis of approximate mass
Two-dimensional electrophoresis is a combination of which two techniques
isoelectric focusing and SDS-PAGE
Which conditions could cause changes in the proteome of a cell?
the developmental stage, the environmental condition, enzymatic modification
Why is an assay necessary for protein-purification studies
an assay allows researchers to accurately measure the amount of the desired protein. This is important in determining if particular purification steps are effective in isolating the protein from the other cellular material.
Compare and contrast gel filtration and ion-exchange chromatography
Although both are used in purification, the properties of the column material determine how the separation is accomplished. Gel filtration is based on porous beads, and molecules are separated by size. In ion-exchange chromatography, the column material is changed with either positively or negatively charged molecules. Separation is base don the proteins’s charge and affinity for the column media.
What is the purpose of determining the specific activity, yield, and purification level of a protein-purification protocol
The measurements allow one to determine whether the individual steps were effective at selectively isolating the protein while maintaining its presence and activity. In order to successfully purify protein, both the yield and the purification level must remain high.
List four possible steps in a protein purification strategy. mention the reason for each.
Differential centrifugation: Separating the cellular components by size
Salting Out: Separating proteins on the basis of their solubility
Gel Filtration Chromatography: Separating proteins based on size
Ion-Exchange Chromatography: separating proteins based on overall charge
Affinity chromatography: separating proteins on the basis of an interaction with a specific chemical.
A protein that on an SDS-PAGE runs as a single 25kilodalton band runs as a 75kilodalton band on a native gel (or gel filtration). Why?
The protein is really a trimer (3 subunits) of 25kDa which gives a native molecular weight of 75 kDa. On an SDS-page, all quaternary structure interactions will be disrupted and it will run with an apparent molecular weight of 25 kDa. However, when the trimer is intact for a native gel or gel filtration column, it runs at 75 kDa
You perform the separation of a protein mixture (ABC) on an ion-exchange column with a buffer at pH 7.4 and get the following elution order: ProteinC, ProteinA, ProteinB. You perform a separation of the same mixture using the same column using a buffer with pH 5.6. The order of elution changes to ProteinA, ProteinC, ProteinB. Why?
The pH of the buffer solution passed over the ion-exchange chromatography has changed between the two experiments. The PH of the solution can modulate the overall charge of the proteins in the mixture and alter the elution profile of the proteins from the ion-exchange column.
What are the advantages of the HPLC over the other chromatographies discussed in chapter 5?
The HPLC method offer higher resolution and faster run times because the resin is made of finer beads and the high pressure allows for faster elution.
Explain three reasons why it is important to know the primary structure of a protein.
- Primary structures from different proteins can be compared to infer knowledge about structure and function. .
- Primary structure comparison of similar proteins from different species provides information about evolution.
- Primary structure can be searched for internal repeats that may yield information on the history of the individual protein.
- Primary structure can reveal the presence of amino acid sequences that regulate protein function and location.
- Primary structure can provide insight into the molecular basis of disease.
- Primary structure can be used as a guide to explore nucleic acid information.
What are two methods for identifying the amino acid sequence of a protein?
Edman degradation and mass spectrometry techniques
the site on the enzyme where the reaction occurs
active site
the substance that the enzyme binds and converts to a product
substrate
enzyme that do not have the required cofactor bound
apenzymes
enzymes will decrease the energy of activation but do not change the _______ of a chemical reaction
delta G or Equilibrium