Biochem chapter 3
Select the true statements about SDS‑PAGE, a method of separating proteins. Assume that SDS‑PAGE is performed under reducing conditions.
-Protein-SDS complexes have similar mass to charge rations; therefore, separation is by size -Smaller proteins migrate faster through the polyacrylamide gel -Proteins are separated in a polyacrylamide gel matrix
SDS-PAGE
-Proteins are separated in their denatured state -Smaller proteins travel faster during separation -proteins move towards the positive electrode
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
ELISA
-proteins are in their native state -can be used to detect antigen or antibody in a sample, depending on procedure
Isoelectric focusing
-proteins are separated by charge -carried out in a pH gradient -final net protein charge is zero
SDS-PAGE
-proteins are separated by mass -carried out in a detergent solution -final net protein charge is negative
Size-exclusion chromatography
-proteins are separated in their native state -Larger proteins travel faster during separation -proteins of similar molecular weight but different shape can be separated
Western blot with SDS-PAGE
-proteins have no higher order structure -proteins transferred to a membrane or sheet
What are the roles of sodium dodecyl sulfate (SDS) in two‑dimensional electrophoresis?
-to cause bound protein to have a large negative charge -to denature proteins
Order the peptides based on their relative positions in the immobilized pH gradient strip at the end of the experiment.
Based on their charges The more positive charged amino acids will have a higher pH and vis a versa
The results of a separation using two-dimension gel electrophoresis are shown here.
Higher pH gives higher pI Larger proteins move slower through the electrophoresis gel
Consider a mixture of four proteins with various molecular weights. A histone molecule weighs 15 kDa,15 kDa, a p53 molecule weighs 53 kDa,53 kDa, an actin molecule weighs 42 kDa,42 kDa, and an IgG molecule weighs 150 kDa.150 kDa. Arrange the molecules in order of their elution from a gel filtration column.
Largest elute first Smallest elute last
Many scientific methods are used to determine protein mass. Which of the following methods would yield the most precise estimate of protein mass?
Mass spectrometry
How do monoclonal antibodies differ from polyclonal antibodies?
Monoclonal antibodies recognize only a single portion of an antigen molecule, whereas polyclonal antibodies recognize different parts of the antigen molecule.
Match each gel pattern to an experimental method. The first lane in each gel is a molecular weight ladder.
Native PAGE- 1 Non-reducing SDS-PAGE- 2 Reducing SDS-PAGE- 3 X polypeptides must =Non-reducing SDS-PAGE Y is other two bars on Reducing SDS PAGE
Consider an experiment where the researcher used Western blotting to detect protein X, which weighs 50 kDa in the whole cell lysates of normal cells and cancer cells. Why are there two bands present in one of the lanes?
Protein X is post-translationally modified in cancer cells
ion exchange chromatography
Separates molecules by charge The stationary phase may contain negatively or positively charged groups
Size-exlusion Chromatography
Separates molecules by size The stationary phase contains cross-linked polymers with different pore sizes
Why are tyrosine kinases other than BTK present in the eluate?
The kinase inhibitor has low specificity
Estimate the molecular weight (𝑀r)(Mr) of the unknown protein that is circled on the graph. Enter the weight as a whole number.
Use the log of the molecular weight to calculate the molecular weight. This requires calculating the antilog (inverse log). log(𝑀r)104.7𝑀r=4.7=𝑀r=50118
Which of the following statements accurately represents characteristics of NMR and x‑ray crystallography?
X‑ray crystallography generally gives a more detailed (higher resolution) view than does NMR.
Which of the following is the LEAST useful for determining the three‑dimensional structure of a protein?
analytical ultracentrifugation
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
Which of the following can SDS polyacrylamide electrophoresis be used to do?
determine the molecular weights of subunits of an oligomeric protein
Select the most appropriate technique for separating the digestion products of AVGWRVKS if cleaved by trypsin.
gel-filtration chromatography
Select one combination of techniques that can be used to isolate Protein B from Proteins A, C, and D.
ion exchange chromatography and gel filtration chromatography
Select the most appropriate technique for separating the digestion products of AVGWRVKS if cleaved by chymotrypsin.
ion-exchange chromatophraphy
Breaking disulfide bonds is a necessary step in preparing proteins for sequencing. Which of the following reagents is used to accomplish this?
mercaptoethanol
A protein has been eluted from a diethylaminoethylcellulose (DEAE‑cellulose) column by washing the column with a buffered salt solution. Since salt can interfere with the next step of your protocol, which is protein quantification, this salt must be removed from your eluted sample.Which of the following will NOT efficiently remove salt from your sample?
passage through a carboxylmethyl-cellulose column
The use of phenyl isothiocyanate, the key reagent in the Edman degradation, makes it possible to
remove and identify one amino acid at a time.
ELISA and Western blot with SDS-PAGE
requires formation of an antigen-antibody complex
Identify the applications for gel filtration chromatography.
separation of components in a mixture by size estimation of molecular weights
A nuclear magnetic resonance, or NMR, spectrometer is an instrument that can be used to characterize molecules based upon
the behavior of the nuclei of certain atoms in a magnetic field
All column chromotography
uses a mobile phase and a stationary phase to separate proteins
