flashcard 12
(50 cards)
What is electrophoresis?
Electrophoresis is the separation of charged molecules under an electric field, causing them to migrate towards the electrode of opposite charge.
Why is Ohm’s law (V = I·R) relevant to electrophoresis?
Because applying a constant voltage increases current as resistance drops, leading to heating; buffers and controlled power supplies prevent the gel from overheating and melting.
What happens if electrophoresis is run without buffer?
Resistance drops, current rises, excessive heat is generated, and the gel may melt or run off the buffer rails.
How do molecule size and charge affect migration speed in a gel?
Smaller and/or more highly charged molecules move faster through the gel matrix, as they experience less hindrance and have higher mobility.
What distinguishes agarose gels from polyacrylamide gels in terms of pore size and typical use?
Agarose gels have larger pores (0.5–2%) suitable for separating larger nucleic acids (DNA/RNA), while polyacrylamide gels have smaller pores for high-resolution separation of proteins and small nucleic acids.
What is the primary use of agarose gel electrophoresis?
To separate DNA fragments in their native (undernatured) form without altering sample characteristics.
What loading dye is commonly used in agarose or polyacrylamide electrophoresis, and what are its functions?
Bromophenol blue (and sometimes xylene cyanol): it adds density to the sample so it settles in the well and provides a visible front to monitor migration.
Why must you monitor gel runs and not leave them unattended?
Because without supervision, samples can migrate off the end of the gel into the buffer, resulting in loss of data.
How are nucleic acids visualized after agarose electrophoresis?
By staining the gel with ethidium bromide (intercalating dye) or non-toxic fluorescent stains, then visualizing under UV light.
What is a Southern blot, and why is it performed?
A Southern blot transfers DNA fragments from an agarose gel onto a nitrocellulose membrane via capillary action; it is used to hybridize labeled probes for sequence‐specific detection (e.g., DNA fingerprinting).
Describe the steps of a Southern blot.
Place the gel on a wick over a buffer reservoir, overlay a nitrocellulose membrane, then paper towels; buffer carries DNA up into the membrane, which is then hybridized with a radioactive or labeled probe and visualized on X-ray film.
What is SDS, and why is it used in SDS-PAGE?
Sodium dodecyl sulfate (SDS) is a detergent that denatures proteins by coating them with uniform negative charge and unfolding them into linear chains.
What is the purpose of boiling protein samples with SDS and β-mercaptoethanol (β-ME)?
Boiling with SDS and β-ME denatures proteins by disrupting secondary/tertiary structure and reducing disulfide bonds, ensuring proteins migrate based solely on size.
What are reducing and non-reducing SDS-PAGE gels?
Reducing gels contain β-mercaptoethanol to break disulfide bonds; non-reducing gels omit β-mercaptoethanol, preserving intramolecular/disulfide linkages and altering migration patterns.
Why are discontinuous buffer systems used in SDS-PAGE?
To create stacking and resolving gels with different pH and ionic strength: the stacking gel concentrates samples into a thin band, then the resolving gel separates proteins by size.
What is the role of the stacking gel in SDS-PAGE?
The stacking gel (higher pH/low acrylamide concentration) focuses proteins into a narrow band before they enter the resolving gel, improving resolution.
How is polyacrylamide gel formed?
Acrylamide and bisacrylamide are polymerized using TEMED (tetramethylethylenediamine) and ammonium persulfate to create a crosslinked matrix.
How are proteins visualized after SDS-PAGE?
By staining the gel with Coomassie Brilliant Blue or non-toxic fluorescent stains to reveal bands corresponding to protein molecular weights.
How is molecular weight estimated on an SDS-PAGE gel?
By running molecular weight standards (markers) alongside samples and comparing migration distances of unknown bands to known marker sizes.
What is Western blotting, and why is it performed?
Western blotting transfers proteins from an SDS-PAGE gel onto a stabilizing membrane (nitrocellulose or PVDF), allowing probing with specific antibodies to detect individual proteins.
Describe the basic steps of a Western blot.
Transfer proteins from gel to membrane, block non-specific sites with protein (e.g., milk powder), incubate with primary antibody, wash, incubate with labeled secondary antibody, and visualize signal to detect target protein.
Why are primary and secondary antibodies used in Western blotting?
The primary antibody binds the specific protein of interest; the secondary antibody binds the primary and carries a reporter/enzyme for signal amplification.
What is isoelectric focusing (IEF)?
IEF separates proteins based on their isoelectric point (pI) by running them in a gel with an immobilized pH gradient; each protein migrates until its net charge is zero (pH = pI).
How is an IEF gel gradient established?
A gel strip is imbued with immobilized pH gradients (e.g., pH 3–9), and proteins are applied at one end; under an electric field, proteins stop migrating at their pI.
