SDS-PAGE resolves polypeptides based on differences in
The mass of a protein as determined by gel filtration is 270 kDa. SDS-PAGE analysis of this protein could produce:
A number of proteins that add up to 270 kDa
Two methods for estimating the size of proteins
SDS-PAGE and gel filtration
SDS-PAGE stands for:
Sodium dodecyl sulfate - polyacrylamide gel electrophoresis
Gel filgration estimates the size of proteins by:
provides as estimate of the molecular weight of the protein in its 'native' state, ie its intact and functional form
What does SDS-PAGE do?
It is the most common type of electrophoresis applied to proteins
It estimates molecular weight by comparing the migration of the protein in its fully denatured or unfolded stated to that of other standard proteins that are also fully denatured.
What breaks disulfide bonds?
A reducing agent, such as β-mercaptoethanol.
It adds a hydrogen across bonds
What are the functions of SDS in SDS-PAGE?
anionic detergent sodium dodecyl sulfate
denature protein, disrupt the noncovalent bonds b/t subunits & cause chain to unfold
produce (-) charge in proportion to protein length
eliminate effect of shape on the determination of polypeptide size
A method of separating charged molecules, such as proteins and nucleic acids, in an electrical field.
What does the rate of a molecule's migration through the electric field in electrophoresis depend on?
the net charge of the molecule
the size of the molecule
the shape of the molecule
the strength of the electric field
Electrophoresis run is completed when fastest migrating protein reaches the bottom of the gel. Is this the smallest or biggest?
How can the proteins be visualized in the electrophoresis?
1. Coomassie blue
2. FITC - fluorescent tag
Polypeptides with greater mass migrate more slowly through the gel matrix in SDS-PAGE because:
They have the same charge:mass ratio as smaller polypeptides and, consequently, their migration is retarded only by their larger size
Why is OD420 used to assay β-galactosidase activity?
o-nitrophenol absorbs light at 420 nm.
What does the enzyme β-galactosidase do?
Catalyzes the hydrolysis of lactose to galactose and glucose
How is enzyme activity measured?
By biochemical assay
What is an assay?
A method to indirectly assess how much of a given protein is produced per cell, per unit time
What does ONPG stand for?
Why is ONPG used?
ONPG is a derivative of lactose. When β-galactosidase cleaves ONPG, it produces galactose and a yellow compound called o-nitrophenol. Thus, ONPG is a useful chromogenic substrate for assaying β-galactosidase activity.
How is absorption measured?
By optical density OD in a spectrophotometer
In the β-galactosidase assay, units of enzyme activity are:
amount of β-galactosidase produced per E. coli cell per minute
In the β-galactosidase assay, cleavage of ONPG by β-galactosidase was terminated by:
adding sodium carbonate (Na2CO3) - changes pH from 7.0 to 11.0
Theoretically, β-galactosidase activity can be assessed by:
measuring lactose consumption
Function of luria broth in biochemical assay:
provides essential nutrients for bacterial growth
Function of Chloroform in biochemical assay:
partially disrupts the cell brane, allowing cellular proteins to diffuse out of the cell
Function of Z-buffer in biochemical assay:
promotes the reaction of β-galactosidase and ONPG by optimizing the pH of the sample
How many bands on a SDS-PAGE gel treaded with β-mercaptoethanol would you see if the protein is a heterodimer?
The Lac Operon regulates bacterial transcription by:
Using lactose to bind to the receptor causing an allosteric change and allowing transcription.
What is required for PCR?
2 primers that bind to denatured genomic DNA at complementary sequences
What is the purpose of Chelex in the DNA isolation procedure?
it tightly binds all the Mg2+ in the extract to prevent DNA degradation
What is Ori H?
One of the origins of DNA replication
In the D-loop region (control region)
What are the two start sites for transcription?
HSP (heavy strand promoter) and LSP (light strand promoter)
In the control region
What does PCR stand for?
Polymerase Chain Reaction
What is PCR?
an in vitro synthesis reaction repeated many times in order to amply DNA using taq-polymerase that catalyzes addition of deoxyribonucleotides and two primers that bind to denatured DNA. Raise temp to stop reaction and lower to let primers hybridize
What does the sequencing primer do?
increases specificity of the product by annealing to a region internal to what was amplified
Rules for primer design
20-30 bp long
G and C 50-60% of total bp
end in a G, C, CG, or GC
Tms b/t 55-80°C
no long runs
How do we use electrophoresis to provide an assessment of DNA quality?
Confirms presence of purified DNA visually and gives qualitative overview of DNA purity (relative intensity compared with markers) and size can be determined based on standard curve generated by markers
gel medium used in DNA lab
proteins are separated by charge in agarose because the pores of the gel are too large to sieve proteins
How does charge of protein and DNA affect which kind of gel is used?
DNA has a uniform charge to mass ratio while polypeptides must be treated with SDS in order to confer a uniform ratio
What is ethidium bromide?
Used in agarose gel as a fluorescent tag
intercalates between DNA base pairs
used to visualize DNA
What was the fluorescent tag used in the polypeptide lab?
What do the spectrophotometry measurements 260nm and 280nm mean?
260nm - DNA absorbs light at this wavelength
280nm - proteins absorb light at this wavelenth
If a ligation reaction is performed attempting to insert DNA fragments into a plasmid vector designed for X-gal screening, and the bacterial colonies are blue, that means:
they are bacterial colonies that have taken up a plasmid wihout an insert
What is the ideal absorption ratio for good nucleic acid purity?
greater than 1.8 ratio of 260nm/280nm (DNA to protein ratio)
What does MRCA stand for?
most recent common ancestor
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