Biotechniques SDS Page and HPLC

What does HPLC stand for?

High-Performance Liquid Chromatography

What is the basic principle of HPLC?

Separation of components in a mixture based on their interactions with the stationary and mobile phases.

What are the main components of an HPLC system?

Solvent reservoir, pump, injector, column, detector, and data system.

What are the two main types of HPLC?

Normal-phase HPLC and reverse-phase HPLC (RP-HPLC).

What is the difference between normal-phase and reverse-phase HPLC?

Normal-phase HPLC uses a polar stationary phase and a non-polar mobile phase, while reverse-phase HPLC uses a non-polar stationary phase (e.g., C18) and a polar mobile phase.

What is the role of the stationary phase in HPLC?

It helps separate compounds based on their interactions with the phase, affecting retention time.

What is retention time (t₀ and tR) in HPLC?

The time it takes for a compound to travel through the column to the detector. t₀ is the time for an unretained compound, while tR is for a retained compound.

What factors affect retention time in HPLC?

Mobile phase composition, column type, temperature, flow rate, and sample characteristics.

What types of detectors are commonly used in HPLC?

UV-Vis detector, fluorescence detector, refractive index (RI) detector, and mass spectrometry (MS) detector.

How does a UV-Vis detector work in HPLC?

It measures absorbance of UV or visible light by analytes as they elute from the column.

What is gradient elution in HPLC?

A technique where the composition of the mobile phase changes over time to improve separation of compounds with varying polarities.

What is isocratic elution?

A method where the mobile phase composition remains constant throughout the run.

What are the advantages of HPLC over traditional column chromatography?

Higher resolution, faster analysis, better reproducibility, and automation.

What is peak broadening in HPLC, and what causes it?

It is the spreading of peaks in a chromatogram, caused by factors like column inefficiency, slow mass transfer, and excessive tubing volume.

What is the purpose of the pump in HPLC?

It delivers the mobile phase at a consistent flow rate through the system.

What role does the injector play in HPLC?

It introduces the sample into the mobile phase before entering the column.

What is the function of the column in HPLC?

It serves as the core separation unit where analytes interact with the stationary phase.

How does temperature affect HPLC separation?

Higher temperatures can reduce viscosity, improve flow, and decrease retention time, but may affect analyte stability.

What is a chromatogram in HPLC?

A graphical representation of detector response versus time, showing the separated components as peaks.

What is the purpose of an internal standard in HPLC?

To improve quantification accuracy by compensating for variations in sample preparation and injection volume.

purpose of the mobile phase in HPLC

transports analytes through the column, controls separation by modulating interactions with the stationary phase, and influences retention time, resolution, and detection efficiency.

What does SDS-PAGE stand for?

Sodium Dodecyl Sulfate - Polyacrylamide Gel Electrophoresis

What is the main purpose of SDS-PAGE?

To separate proteins based on their molecular weight.

What is the role of SDS (Sodium Dodecyl Sulfate) in SDS-PAGE?

SDS is a detergent that denatures proteins by disrupting non-covalent bonds and giving proteins a uniform negative charge. overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure

What is the function of polyacrylamide in SDS-PAGE?

It forms a gel matrix that acts as a molecular sieve, separating proteins based on size.

What is the role of β-mercaptoethanol (BME) or Dithiothreitol (DTT) in SDS-PAGE?

hey reduce disulfide bonds, further denaturing proteins and breaking quaternary structures.

What is the purpose of the stacking gel in SDS-PAGE?

It concentrates proteins into a narrow band before they enter the resolving gel, improving resolution.

What is the purpose of the resolving gel in SDS-PAGE?

It separates proteins based on size as they migrate through the gel matrix.

What is the relationship between protein size and migration distance in SDS-PAGE?

Smaller proteins migrate faster and travel farther, while larger proteins move more slowly.

What is the function of the loading buffer in SDS-PAGE?

It contains SDS, a reducing agent, and tracking dye to help visualize sample loading and migration.

What is the purpose of Coomassie Brilliant Blue or Silver Staining in SDS-PAGE?

o visualize proteins after electrophoresis by binding to them and making them visible.

What is the role of the molecular weight marker (protein ladder) in SDS-PAGE?

It provides reference bands of known molecular weights to estimate the sizes of unknown proteins.

Why is a constant voltage or current applied during SDS-PAGE?

To drive negatively charged proteins through the gel towards the positive electrode (anode).

How does acrylamide concentration affect protein separation in SDS-PAGE?

Higher acrylamide concentrations resolve smaller proteins better, while lower concentrations are better for larger proteins.

What is the role of ammonium persulfate (APS) and TEMED in SDS-PAGE?

They catalyze the polymerization of acrylamide to form the gel.

What is Western blotting, and how is it related to SDS-PAGE?

Western blotting is a technique that transfers proteins from an SDS-PAGE gel to a membrane for detection using antibodies.

What does "denaturing" mean in the context of SDS-PAGE?

It refers to the unfolding of proteins into linear chains, eliminating secondary and tertiary structures.

What effect does glycerol have in the loading buffer?

It increases the sample density, helping it sink into the wells during loading.

What happens if the gel is run for too long?

The proteins may run off the gel or become too diffused, reducing resolution.

What is the difference between native PAGE and SDS-PAGE?

Native PAGE separates proteins based on size, charge, and shape, while SDS-PAGE separates only by size.

Why is Tris buffer used in SDS-PAGE?

Tris maintains a stable pH during electrophoresis, preventing protein denaturation or pH shifts.

Retention Factor (k')

(tR - t0) / t0

Why does SDS-treated protein migration depend only on molecular weight?

DS imparts a uniform charge-to-mass ratio, eliminating differences in shape and charge.

Why is a constant charge-to-mass ratio important in SDS-PAGE?

It ensures that proteins are separated purely based on molecular weight rather than charge or shape.

What is the purpose of boiling protein samples before loading them into an SDS-PAGE gel?

To fully denature the proteins, allowing SDS to bind uniformly.

What is the composition of a stacking gel, and what is its role?

0.125 M Tris-HCl (pH 6.8) with 5% acrylamide; it concentrates proteins into a narrow band before entering the resolving gel.

What is the composition of a resolving gel, and what is its role?

0.375 M Tris-HCl (pH 8.8) with 12% acrylamide; it separates proteins based on molecular weight.

What is the importance of glycine ionization in SDS-PAGE?

The change in glycine charge between the stacking and running gels facilitates protein separation.

Why is polyacrylamide gel considered a "crosslinked matrix"?

It consists of polymerized acrylamide monomers, creating a stable sieving structure.

What safety precautions should be taken when working with unreacted acrylamide?

Acrylamide monomers are neurotoxic, so proper handling and disposal are essential.

How does protein migration differ in the stacking and resolving gels?

Proteins migrate quickly in the stacking gel and slow down in the resolving gel, where they separate by size.

What are the key principles governing protein separation in SDS-PAGE?

Uniform charge-to-mass ratio, molecular sieving by gel matrix, and migration in an electric field.

What happens to proteins after SDS-PAGE?

They are stained (e.g., Coomassie Blue or Silver Stain) for visualization or transferred to a membrane for Western blotting.

What is the role of free radicals in gel polymerization?

They initiate the vinyl polymerization reaction that forms the polyacrylamide gel.

What charge do SDS-coated proteins have, and where do they migrate?

They have a negative charge and migrate toward the anode (+) in the electric field.