Key Concepts in Chromatography and Protein Analysis

Imidazole in His-tag affinity chromatography

It competes with the His-tag to bind Ni²⁺, eluting the protein from the resin.

Ni-NTA

Nickel-nitrilotriacetic acid, used to bind His-tagged proteins.

Four main types of liquid chromatography

Affinity, Ion Exchange, Size Exclusion, and Hydrophobic Interaction Chromatography.

SEC

Size Exclusion Chromatography.

SEC elution order

Large proteins elute first because they cannot enter the pores.

Isoelectric point (pI)

The pH at which a protein has no net charge.

Cation exchange chromatography column charge

Negative charge (it binds positively charged proteins).

Kav in SEC

A partition coefficient that describes elution volume: Kav = (Ve - Vo)/(Vt - Vo).

MALDI-TOF ionization type

Laser desorption/ionization with a matrix that absorbs UV light.

MALDI-TOF detector order

Small ions, because they move faster at the same kinetic energy.

TOF

Time of Flight.

Enzyme for protein digestion before mass spectrometry

Trypsin.

Trypsin cleavage specificity

The carboxyl side of arginine (R) and lysine (K).

Tandem MS purpose

To sequence peptides by fragmenting them and analyzing the resulting ions.

Bradford assay wavelength

595 nm.

Bradford assay color change cause

Coomassie dye shifts to an anionic state when bound to protein.

Beer-Lambert Law

A = εbc, where A is absorbance, ε is molar absorptivity, b is path length, and c is concentration.

Amino acids that absorb at 280 nm

Tryptophan and Tyrosine.

ε for Tryptophan at 280 nm

5500 M⁻¹cm⁻¹.

Michaelis-Menten equation

v₀ = (Vmax [S]) / (Km + [S]).

Vmax

The maximum rate of the reaction when the enzyme is saturated.

Km

The substrate concentration at which the reaction rate is half of Vmax.

Low Km indication

High substrate affinity.

kcat

Turnover number: number of substrate molecules converted per enzyme per unit time.

Catalytic efficiency calculation

kcat / Km.

Competitive inhibition effect on Km and Vmax

Km increases, Vmax stays the same.

Uncompetitive inhibition effect on Km and Vmax

Both Km and Vmax decrease.

Mixed inhibition effect on Km and Vmax

Vmax decreases, Km may increase or decrease.

Secondary structure of proteins

Alpha helices and beta sheets.

Anfinsen's principle

The amino acid sequence determines protein structure.

Most used technique in PDB for protein structure

X-ray crystallography.

Bragg's Law

nλ = 2dsinθ, relates X-ray diffraction to crystal structure.

Small 'd' value in Bragg's Law indication

High resolution (closely spaced atomic planes).

X-rays in crystallography reason

Their wavelength is similar to the length of chemical bonds.

Three phases of protein X-ray crystallography

Crystal growth, data collection, and model building.

Asymmetric unit

The smallest unit of a crystal structure that can build the entire crystal by symmetry.

Three steps of PCR

Denaturation, annealing, and extension.

Denaturation

The process of separating DNA strands by heating.

Annealing

The step in PCR where primers bind to the target DNA sequence.

Extension

The phase in PCR where DNA polymerase synthesizes new DNA strands.

Typical temperature for denaturation in PCR

95°C.

Key components needed for PCR

Template DNA, DNA polymerase, forward primer, reverse primer, dNTPs, Mg²⁺.

Tm (melting temperature) of a primer

Tm ≈ 2°C(A+T) + 4°C(G+C).

Site-directed mutagenesis

A technique used to introduce specific mutations into a DNA sequence.

Strain typing by PCR

Using primers that target variable regions to identify bacterial strains.

RT-qPCR

A method that detects RNA viruses by converting RNA to DNA and amplifying it.

SARS-CoV-2 RT-qPCR

Primers target the N gene, RNA is reverse transcribed to DNA, and fluorescence signals presence.

Molecular cloning

Cutting and pasting DNA fragments into plasmids for replication or expression.

Key features of a plasmid used in cloning

Origin of replication, selectable marker, and multiple cloning site.

Restriction endonucleases

Enzymes used to cut DNA at specific sequences.

Role of DNA ligase in cloning

It seals the DNA backbone, joining sticky or blunt ends.

Directional cloning

Using two different restriction enzymes to insert DNA in a specific orientation.

Ampicillin resistance in cloning

It serves as a selectable marker to identify transformed cells.

Lac repressor

It binds the operator and blocks transcription in the absence of allolactose.

Effect of allolactose presence

It binds the repressor, allowing RNA polymerase to transcribe the operon.

Shine-Dalgarno sequence

A ribosome binding site in bacterial mRNA.

Types of transcription termination

Intrinsic (hairpin + U's) and Rho-dependent (uses Rho protein).

Anfinsen's experiment

Showed that protein folding is determined by its amino acid sequence.

Ramachandran plot

A graph of allowed phi and psi angles in a polypeptide.

Stabilization of alpha helices and beta sheets

Hydrogen bonds between backbone atoms.

Residues that disrupt alpha helices

Proline and glycine.

Rise per turn in an alpha helix

5.4 Å per turn with 3.6 residues per turn.

Requirement for X-ray diffraction

A well-ordered protein crystal.

Bragg's Law

Describes the condition for constructive interference of X-rays.

Mirror or inversion symmetry in protein crystals

Cannot apply because proteins are made of L-amino acids and are chiral.

Screw axis

A symmetry operation combining rotation and translation.

Modeling phase of crystallography

Building a 3D structure into the electron density map.

NDA reaction in enzyme assay for SufS

Forms a fluorescent conjugate with alanine, allowing quantification.

High kcat/Km ratio

Indicates a highly efficient enzyme.

Endpoint assay

An assay that measures product after a fixed time.

Steady-state kinetics

Analysis of enzyme behavior under conditions where [ES] is constant.

Function of Mg²⁺ in PCR

It is a cofactor required by DNA polymerase for activity.

Primers length in PCR

Usually 15-30 bases long to ensure specificity and strong binding.

GC clamp in primer design

One or more G/C bases at the 3' end to increase stability.

G/C bases at the 3' end

One or more G/C bases at the 3' end to increase stability.

Forward and reverse primers in PCR

To amplify both strands of the DNA template.

Uracil-N-glycosylase in RT-qPCR

To prevent contamination by degrading uracil-containing DNA.

Reverse transcription

It converts RNA to DNA before amplification.

Quantitative nature of RT-qPCR

Fluorescent signal increases proportionally with DNA amount.

Selectable marker

A gene that allows identification of cells with a plasmid, e.g., antibiotic resistance.

MCS

Multiple Cloning Site.

Restriction site within gene of interest

The gene might be cut and not fully cloned.

Importance of plasmid orientation in cloning

Correct orientation is needed for proper gene expression.

Transformation in molecular biology

Introducing foreign DNA into a cell.

Sanger sequencing

It uses dideoxynucleotides to terminate DNA synthesis at specific bases.

Termination of DNA synthesis by ddNTPs

They lack a 3' OH group required for chain elongation.

Analysis of Sanger sequencing products

By capillary electrophoresis and fluorescence detection.

Advantage of next-generation sequencing (NGS)

It allows parallel sequencing of millions of fragments.

Function of a flow cell in Illumina sequencing

It immobilizes DNA fragments for amplification and sequencing.

m/z

Mass-to-charge ratio.

Use of trypsin before MS analysis

It generates predictable peptide fragments.

First step in MALDI-TOF

Laser desorption and ionization of the sample.

Molecules best analyzed by MALDI-TOF

Proteins and peptides.

Role of the matrix in MALDI

It absorbs laser energy and transfers it to the analyte.

Function of TCEP in enzyme assays

It reduces disulfide bonds to keep proteins in a reduced state.

Use of NDA in fluorescence-based enzyme assays

It reacts with alanine to form a fluorescent product.

Alanine fluorescence in the SufS assay

Alanine is a product of the desulfurase reaction.

Continuous assay

One that tracks reaction progress in real time.

Discontinuous assay

One that requires stopping the reaction at intervals.

Primary protein structure

The linear sequence of amino acids.