Biochemistry Chapter 3 Homework
Select the true statements about SDS‑PAGE, a method of separating proteins. Assume that SDS‑PAGE is performed under reducing conditions.
-Proteins are separated in a polyacrylamide gel matrix. -Protein‑SDS complexes migrate toward the negative electrode. -Smaller proteins migrate faster through the polyacrylamide gel. -Sodium dodecyl sulfate binds proteins, resulting in protein‑SDS complexes that are similar in size. -Proteins are visualized using a dye that binds to the gel matrix, but not to proteins. -A protein binds roughly 1.4 times its mass of SDS, resulting in a large overall negative charge.
An IgG antibody that specifically recognizes a protein of interest, p50, was used to isolate p50 from a whole‑cell lysate. The resulting protein mixture can undergo SDS-PAGE to separate p50 from IgG. The molecular weight of p50 is 50 kDa, and the protein does not contain any cysteine residues. The molecular weight of IgG is 160 kDa. IgG contains two heavy chains and two light chains that weigh 50 kDa and 25 kDa, respectively. Both types of chains contain cysteine residues. Select the components of the SDS‑PAGE loading buffer.
-glycerol -sodium dodecyl sulfate -bromophenol blue
Select all the parameters that can be determined by analyzing a protein sample with tandem mass spectrometry.
-precursor ion mass -amino acid sequence
What are the characteristics of a secondary antibody used in Western blotting?
-recognizes the Fc region of the primary antibody -contains a covalently attached tag
Identify the applications for gel filtration chromatography.
-separation of components in a mixture by size -estimation of molecular weights
What characteristics determine the position of a protein on an IPG strip at the end of isoelectric focusing?
-the pI of the protein -local pH in the medium
What are the roles of sodium dodecyl sulfate (SDS) in two‑dimensional electrophoresis?
-to denature proteins -to cause bound proteins to have a large negative charge
Upon centrifugation, particles sediment at different rates due to their physical properties. Tropomyosin is a rod-shaped protein with a mass of 70 kDa and a sedimentation coefficient of 2.6S. Hemoglobin is a spherical protein with a slightly smaller mass of 65 kDa, but a much higher sedimentation coefficient of 4.31S. Which property of tropomyosin accounts for its slow sedimentation?
Elongated shape
A researcher resolves a mixture of peptides using isoelectric focusing. Order the peptides based on their relative positions in the immobilized pH gradient strip at the end of the experiment.
LowpH Glu-Gly-Glu-Asp Asp-Ala-Leu-Asp Ars-Gly-Glu-Lys Ars-Leu-Ala-Ars Ars-Ala-Lys-Lys HighpH
Each of the given statements describes a type of column chromatography. Match the statements to the type of chromatography they describe. If a statement can describe all of the types, place that statement in the All category. (Note that size‑exclusion chromatography may also be called gel filtration or molecular‑exclusion chromatography.)
Size-Exclusion Chromatography: Separates molecules by size; the stationary phase contains cross-linked polymers with different pore sizes. Affinity Chromatography: Can separate molecules based on protein-ligand binding; The stationary phase has a covalently bound group to which a protein in the mobile phase can bind. Ion-exchange chromatography: Separates molecules by charge; The stationary phase may contain negatively or positively charged groups. All:Uses a mobile phase and stationary phase to separate protein
Consider an experiment where your goal is to isolate Bruton's tyrosine kinase (BTK) enzyme from a whole‑cell lysate. You have an affinity chromatography column with a tyrosine kinase inhibitor molecule covalently attached to the beads. The tyrosine kinase inhibitor binds and inhibits BTK. As a result of the experiment, you are able to elute BTK from the column, but in a mixture of other tyrosine kinases. Why are tyrosine kinases other than BTK present in the eluate?
The kinase inhibitor has low specificity.
Identify the buffer solution that can be used for eluting a transcription factor bound to a DNA affinity column.
high salt concentration