Biomolecules 1B

Isoelectric focusing

Molecular biology technique used to separate molecules (AA, peptides, proteins) based on their pI/pH when it carries no net electric charge

• molecules stop migrating where the pH = pI

Basic/positively charged AA reach neutrality in

In more basic conditions

Acidic/negativly charged AA reach neutrality in

In more acidic conditions

In acidic conditions, the carboxyl group of AA

Becomes protonated

In basic conditons, the amino group of the AA

Becomes deprotonated

Acidic AA has a ____ pI

Lower pI

Basic AA has a ____ pI

Higher pI

Protein electrophoresis

Separates proteins based on size and charge using an electric field through a gel

Native PAGE

Separates proteins without using denaturing agents

SDS-PAGE

Separates proteins based on molecular weight by denaturing the protein and coating with a negative charge

SDS (Sodium Dodecyl Sulfate)

Anionic detergent that binds to and denatures proteins into a linear chain

PAGE

Polyacrylamide Gel Electrophoresis

Applications of SDS-PAGE

Determine molecular weight, analyze protein purity, protein identification, and western blotting

Westren blotting

Technique for protein identification

Steps for western blotting

1. Separation: protein separation by mw (SDS-PAGE)

2. Transfer: proteins transferred from gel to a membrane using electroblotting

3. Staining: target specific protein with an antibody (amplified with a secondary antibody) and block specific binding sites with a blocking buffer

4. Visualization: substrate enzyme added, giving a detectable signal (chemiluminescence/color change) on the target protein

Western blotting applications

Detect specific proteins, measure/quantify levels of specific proteins, detecting post-translational modifications

Enzyme-linked immunoassay (ELIZA)

Technique that used antibodies to detect and quantify proteins directly in a liquid sample (without a transfer)

Southern blotting

Technique used to detect DNA sequences, analyze gene structure and organization, and diagnosis genetic disorders

Steps of southern blotting

1. DNA extraction and digestion: enzymes used to break into fragments

2. Gel electrophoresis: separates fragments

3. Blotting: sample transferred from gel to a membrane

4. Probing: sample incubated with labeled DNA probe binds to specific fragments (hybridized)

5. Detection: detect hybridized DNA-probe complexes with autoradiography, fluorescence, or chemiluminescence

Northern blotting

Technique for RNA — measuring gene expression levels, analyzing RNA processing and stability, and detecting RNA transcripts

Steps of northern blotting

1. RNA isolation: isolate RNA from cells

2. Gel electrophoresis: separate RNA using agarose gel electrophoreses

3. Transfer/blotting: transfer the RNA from the gel to a blotting membrane (maintaining position of fragments)

4. Hybridization: incubate fragments with a labeled probe that binds to/hybridizes to target RNA

5. Washing: excess probe is washed away

6. Detection: detect hybridized RNA-probe complexes (specific to labeled probe)

Chromatography

Separation of molecular components (proteins) based on size, charge, hydrophobicity, binding affinity, and polarity

Mobile phase

“Carrier”; solvent moving through the column

Stationary phase

“adsorbent”; substance that stays fixed inside the column

Normal-phase chromatography

Non-polar solvent and polar stationary

Reverse-phase chromatography

Polar mobile phase and non-polar stationary

Eluent

Fluid entering the column

Eluate

Fluid exiting the column

Elution

The process of washing out a compound through a column using a solvent

Analyte

Mixture whose individual components have to be separated and analyzed

Retention time

Time it takes for a component to pass through the column to be detected

Gradient elution

Technique changing the mobile phase during separation process

Adsorption

How well molecules adhere to a surface

Solubility

Ability of a molecule to dissolve in a solvent

Higher adsorption to stationary phase

Slower/less the molecule moves through the column

Higher solubility in the mobile phase

Faster/more the molecule will move through the column

Retention factor (R_f)

Distance traveled by the component compared to the solvent

component/solvent

Paper chromatography

Compound spotted directly on a cellulose paper

• stationary phase: solid (cellulose)

• mobile phase: liquid

• separation: polarity of molecules

Thin layer chromatography (TLC)

Glass plate is coated with thin layer of the stationary which is spotted with the sample compound

• stationary phase: solid (silica or alumina)

• mobile phase: liquid

• separation: polarity of molecules (most polar moves the least)

Liquid column chromatography

Glass column is packed with the stationary phase and the sample is separated as it moves through

• stationary phase: solid (silica or alumina)

• mobile phase: liquid

• separation: polarity of molecules

Size exclusion chromatography (SEC)

Separate proteins based on size by passing through pores of the stationary phase

• stationary phase: solid (microporous beads of silica)

• mobile phase: liquid

• separation: size of molecules

Ion-exchange chromatography

Separate proteins based on net charge (pH of buffer)

• stationary phase: solid (cationic or anionic resin → binds to proteins of opposite charge)

• mobile phase: liquid

• separation: ionic charge of the molecules

Affinity chromatography

Separate proteins based on their specific interactions w/ a ligand

• stationary phase: solid (agarose or porous glass beads of immobilized enzyme, antibody, inhibitor, or metal ion)

• mobile phase: liquid

• separation: binding affinity of the analyte molecule to the stationary phase

Gas chromatography (GC)

Samples are volatilized and the molecule with lowest boiling point comes out of the column first → highest comes out last

• stationary phase: liquid or solid support

• mobile phase: gas (inert gas like argon or helium)

• separation: boiling point of molecules

High performance liquid chromatography (HPLC)

Separation, identification, and quantification of components using a high pressure pump

Reverse-phase HPLC (RP-HPLC)

Stationary phase is non-polar (hydrophobic) and the mobile phase is polar. It is ideal for separating non-polar and moderately polar compounds

Normal-phase HPLC (NP-HPLC)

Stationary phase is polar, and the mobile phase is non-polar. It is used for separating polar compounds

Ion-exchange HPLC

Utilizes a charged stationary phase to separate ionic compounds based on their charge. It is widely used for proteins, peptides, and other charged biomolecules.

Size-exclusion HPLC (SEC HPLC)

Separates molecules based on their size. Larger molecules elute first because they are excluded from the pores of the stationary phase, while smaller molecules take longer as they penetrate the pores

Affinity HPLC

Employs a stationary phase with specific ligands that can bind selectively to certain molecules, such as proteins or antibodies, allowing for highly specific separations

HPLC detectors

UV-visible, fluorescence, refractive index (RID), mass spectrometer (MS

HPLC applications

Pharmaceuticals, environment analysis, analysis in food industry, clinical diagnosis, biochem/proteomics

Ultracentrifugation

Separates protein based on size, shape, and density using centrifugation (sedimentation)

Sedimentation coefficient

Measurement of how fast particles sediment under the influence of centrifugal force

• measured in Svedberg units “S”

• larger proteins = higher sedimentation coefficient

Ultracentrifugation application

Purify complexes, study molecular weigh and shape, isolate organelles and subcellular structures

Mass spectrometry

Identify and quantifies proteins based on mass-to-charge ratio (m/z)

Mass spectrometry procedure

1. Sample prep: fragment proteins into peptides using enzymes

2. Ionization (MALDI or ESI)

3. Separation and detection: peptides separated in the spectrometer by their mass to charge ratio (m/z)

4. Data analysis: read by different peaks

Isotope-coded affinity tags (ICAT)

A method that involves labeling proteins with isotopic tags, which allows for quantification and identification of proteins via mass spectrometry

Surface plasmon resonance (SPR)

Measures the interaction between molecules (e.g., protein-protein, protein-ligand) in real time without the need for labeling

Two-dimensional gel electrophoresis (2D-GE)

Combines isoelectric focusing (separation based on isoelectric point) and SDS-PAGE (separation based on size) for high-resolution protein separation

Protein microarrays

Involves immobilizing proteins on a solid surface to analyze interactions, functions, and abundance of proteins

Fourier-transform infrared spectroscopy (FTIR)

Analyzes protein secondary structures based on infrared absorption spectra

Nuclear Magnetic Resonance (NMR)

Provides detailed information on the structure and dynamics of proteins in solution

Circular Dichroism (CD) spectroscopy

Measures the absorption of circularly polarized light to analyze protein secondary structures