Electrophoresis and Spectroscopy

Biochemistry Lecture Notes: Gel Electrophoresis and Spectroscopy

Lecture Details

• Date: 9/5/25

• Instructor: Jon Marles-Wright

• Email: jon.marles-wright1@ncl.ac.uk

• Course: BGM1002

• Lecture Number: 9

Learning Objectives

• By the end of this lecture series, you will be able to answer the following key questions:

  • Why do we study proteins?

  • How do we study proteins?

  • What are proteins?

  • How do proteins work?

Methods for Studying Proteins

• Various scientific disciplines involved include:

  • Biophysical Chemistry

  • Chemistry

  • Biology

  • Physics

  • Mathematics

  • Note: This is a gross simplification of the interdisciplinary nature of protein study.

Electrophoresis Overview

• Definition: Movement of dispersed particles relative to a fluid under the influence of a spatially uniform electric field.

• Separation Capabilities:

  • Proteins

  • Nucleic acids

  • Glycans

• Common Techniques: Utilizes gels or capillaries for separation.

Electrophoresis Mechanism

• Charged species migration:

  • Charged particles (both positive and negative) are influenced by an electric field, leading to movement:

  • Positive charges move towards the negative electrode (+).

  • Negative charges move towards the positive electrode (-).

• A current flow is established through the system during this process.

Gel Electrophoresis

• Mechanism and Composition:

  • Polymer gels serve as molecular sieves, with pore size influenced by the concentration of the gelling agent.

  • Types of Gelling Agents:

  • Agarose:

    • Linear polymer derived from red seaweed, used for nucleic acids/protein complexes.

  • Acrylamide:

    • Organic compound with multiple functional groups, crosslinked using a catalyst, used for proteins/nucleic acids.

  • Buffering: Liquid phases must be buffered to stabilize against current-induced pH changes and minimize heating, which can affect gel performance.

Factors Affecting Gel Electrophoresis

• Variables Influencing Process:

  • Net charge of the molecule

  • Size of the molecule

  • Strength of the electric field

  • Properties of the gel (e.g., concentration)

  • Properties of the running buffer:

  • pH

  • Counter ions

  • Salts

  • Temperature

Electrophoresis Variants

Native PAGE

• Purpose: Separation of acidic proteins by charge and size without denaturing agents.

• Sample Preparation: Samples added to buffer with dye/glycine.

• Benefits: Suitable for studying protein complexes and post-translational modifications.

Blue Native PAGE

• Modification: Addition of Coomassie blue to provide additional charge to proteins, which can lead to dissociation of complexes.

SDS-PAGE

• Definition: Proteins are denatured using heat and sodium dodecyl sulfate (SDS) to form mixed protein:SDS micelles.

• Separation Criterion: Based on mass rather than charge.

• Challenges with Specific Proteins:

  • Some proteins resistant to denaturation (e.g., disulfide bonds require reducing agents, thermostable proteins).

• Operational Details: Running DNA is possible on PAGE gels.

Properties of SDS-PAGE

• Gel Structure: Discontinuous gels created.

  • Stacking Gel: pH 6.5, low % acrylamide for focusing proteins before separation.

  • Resolving Gel: pH 8.8, higher % acrylamide for separation based on size.

  • Changing acrylamide concentration impacts separation resolution.

  • Common buffer components include glycine/chloride counter ions affecting protein migration.

• Gradient Gels: Designed with increasing acrylamide concentration down the gel.

Agarose Gel Electrophoresis

• Composition: 0.5-2% agarose in TAE/TBE buffer.

• Buffer Components:

  • Tris buffer (pH 8.3)

  • Acetate/Borate counter ions

  • EDTA as a chelating agent.

• Separation Ranges: 100 – 500 nm; excellent for DNA ranging from 50 base pairs to 20 kilobases, but with lower resolution compared to PAGE.

Visualizing Molecules in Gels

• Intrinsic Color: DNA and most proteins require staining to be visualized.

• Types of Stains/Dyes and Sensitivities:

  • Nucleic Acids:

  • Ethidium bromide (intercalating dye)

  • Hoechst, DAPI (minor groove binders)

  • SYBR (Cyanine dyes)

  • Protein Gels:

  • Coomassie blue

  • Silver stain

  • Sypro stains with varying sensitivity (fluor orange).

Molecular Weight Estimation

• Comparison of sample migration vs. standard curve generated from protein/nucleic acid standards:

  • Calculation Method:

  • Measure migration distance of standards and dye front.

  • Calculate relative mobility using the formula:
    $Rf = \frac{\text{migration distance of protein}}{\text{migration distance of dye}}$

  • Create a plot of log(MW) of standards versus $Rf$, derive a linear equation to calculate MW of unknowns.

• Modern Techniques: Automatic measurements can be performed using gel imagers for high accuracy.

Common Problems with Gels

• Anomalies and Issues:

  • Smileys: Caused by voltage being too high.

  • Melting gels: Occurs when gels overheat due to excessive voltage.

  • Smearing or contaminant presence leading to poor results.

  • Observational Issues: Bubbles, speckles, or improperly loaded samples, which can be problematic during practical lab sessions.

Spectroscopy Overview

• Definition: Study of the interaction between matter and electromagnetic radiation.

• Applications: Widely used in various fields of chemistry and biochemistry.

Electromagnetic Spectrum

• Various molecular movements governed by electromagnetic radiation include:

  • Molecular Rotations

  • Molecular Vibrations

  • Electron Transitions

UV/Visible Spectroscopy

• Basics: Absorption spectroscopy with light absorption impacting atomic/molecular energy, prompting electronic transitions of various forms

  • Key Transitions:

  • $σ o σ^*$

  • $π o π^*$

  • $n o σ^*$

  • $n o π^*$

  • $ext{aromatic } π o ext{aromatic } π^*$

• Color Perception: Dependent on environmental factors leading to bathochromic (longer wavelength) or hypsochromic (shorter wavelength) shifts.

UV/Vis Absorption Spectroscopy Applications

• Use Cases:

  • Quantification of proteins/nucleic acids

  • Analysis of protein/nucleic acid unfolding

  • Characterization of substrate/cofactor binding to proteins

  • Implementation in biochemical assays.

Measuring Protein Concentration

• Beer-Lambert-Bouguer Law:

  • $A = \epsilon \ell c$ where:

  • $A$ = absorptivity of a substance

  • $\epsilon$ = molar attenuation coefficient

  • $\ell$ = optical path length

  • $c$ = concentration of attenuating species

  • Key Assumptions:

  • Monochromatic/parallel illumination

  • Homogeneous solution

  • No scattering from the medium

  • Linear absorbance change with concentration.

Quantification of Proteins

Direct Measurements (A280)

• Amino Acids that Absorb: Tryptophan, tyrosine, and disulfides absorb light at around 280 nm wavelength.

• Molar Extinction Coefficient Estimation Formula:

  • $\epsilon = (nW \times 5,500) + (nY \times 1,490) + (nC \times 125)$

• Note: If a protein lacks these amino acids, it will not absorb at 280 nm.

• Sensitivity Range: Reasonably sensitive within 50 – 100 μg.

• Example Calculation: For protein example "MAQSSNSTHEPLEVLKEETVNRHRAIVSVMEELEAVDWYDQRVDASTDPELTAILAHNRDEEKEHAAMTLEWLRRNDAKWAEHLRTYLFTEGPITAIEAADTAGEGSGGDAAKGATAQGDSGLGIGSLKGEAALARPPRL"

  • $W: 3, Y: 2, C: 0$
    Molecular Weight: $15192.74$
    Calculation: $\epsilon = (3 \times 5,500) + (2 \times 1,490) + (0 \times 125) = 19480 M^{-1} cm^{-1}$

Indirect Measurements

• Methods Include:

  • Lowry Method

  • Bradford Assay

  • Biuret Method: React proteins with copper sulfate and sodium hydroxide, resulting in a violet color change at A540 nm. Slow response time (20-30 mins) with moderate sensitivity (1-20 mg).

  • BCA Assay: Involves binding of copper to nitrogen in proteins followed by binding to bicinchoninic acid, resulting in a purple color change measured at A562. High sensitivity at 1 μg, response time can be up to 1 hour unless using faster kits.

  • Note: Sensitivity can be affected by nitrogenous contaminants.

Summary of Lecture Content

• Principles of electrophoresis covered including:

  • PAGE and Agarose gel electrophoresis.

  • Methods for estimating size of proteins/DNA via gels.

  • Techniques for staining DNA/proteins in gels.

  • Fundamentals of UV-visible spectroscopy and protein quantification in solution.