Lab Techniques

Gel Electrophoresis

Basic Principle

A technique that separates macromolecules (proteins, DNA, or RNA) based on sizeand/or charge.

How It Works

• Molecules are placed in lanes within a gel

• Gel types:

  • Polyacrylamide: Used for proteins and small DNA/RNA molecules

  • Agarose: Used for larger DNA molecules (>500 base pairs)

• An electrical field is applied across the gel:

  • Anode (+) at the bottom

  • Cathode (-) at the top

• Functions like an electrolytic cell

• Negatively charged molecules migrate toward the positive anode

• Smaller molecules move faster through the gel matrix, so they end up farther down

• A ladder (known-size standards) is run alongside samples for size comparison

• Gels are stained for visualization (commonly Coomassie Blue)

Types of Gel Electrophoresis for Proteins

Native-PAGE

Feature	Description
Conditions	Non-denaturing
What is preserved	Protein structure and function
Separation basis	Size (while maintaining native shape)

SDS-PAGE

Feature	Description
Conditions	Denaturing
Key reagent	SDS (sodium dodecyl sulfate) - negatively charged
How it works	SDS denatures proteins and binds at a ratio of 1 SDS per 2 amino acids
Result	All proteins get the same charge-to-mass ratio
Separation basis	Mass only (smaller = faster)
Limitation	Does NOT break disulfide bridges (covalent bonds)

Memory tip: SDS = "Same charge-to-mass ratio, Denatures, Separates by Size"

Reducing SDS-PAGE

• Same as SDS-PAGE PLUS a reducing agent (e.g., β-mercaptoethanol)

• Breaks disulfide bridges (S-S bonds)

• Results in completely denatured protein (linear shape)

• Useful for studying proteins with multiple subunits

Isoelectric Focusing

Feature	Description
Separation basis	Isoelectric point (pI) - the pH where a protein has zero net charge
Gel feature	Contains a pH gradient
How it works	Proteins migrate until they reach the pH that equals their pI
At pI	Net charge = 0, protein stops moving

Key insight: Proteins with more acidic residues have lower pI; proteins with more basic residues have higher pI.

Western Blotting

Purpose: Detect a specific protein in a sample

Steps:

1. Separate proteins using SDS-PAGE

2. Transfer proteins from gel to a polymer membrane

3. Probe with an antibody specific to the protein of interest (may use primary + radiolabeled secondary antibody)

4. Visualize using autoradiography

Key point: Uses antibodies for protein identification

Southern Blotting (DNA) and Northern Blotting (RNA)

Southern Blotting Steps

Step	Action
1	Cut DNA with restriction enzymes
2	Denature DNA with NaOH → single-stranded DNA
3	Separate fragments by gel electrophoresis
4	Transfer to nitrocellulose membrane
5	Expose to radiolabeled DNA probe (complementary to target)
6	Visualize with autoradiography

Northern vs. Southern Blotting

• Northern blotting uses RNA instead of DNA

• Steps 1 and 2 (restriction digest and denaturation) are NOT performed for Northern blotting

• Everything else is essentially the same

DNA Sequencing (Sanger Method)

Purpose

Determine the exact nucleotide sequence of a DNA strand

Key Concept: Dideoxynucleotides (ddNTPs)

• Modified nucleotides missing the 3' OH group

• Cannot form phosphodiester bonds → chain termination

• Each ddNTP is added to a separate reaction tube

Procedure

Step	Description
1	Denature DNA with NaOH → single-stranded template
2	Set up 4 separate reactions, each containing: DNA template, radiolabeled primer, DNA polymerase, all 4 dNTPs, and ONE type of ddNTP (small amount)
3	Run each reaction in its own lane on a gel
4	Read the sequence bottom to top (smallest fragments = closest to primer)

Reading the Gel

• Each band represents a fragment that terminated at a specific nucleotide

• Read from bottom (5' end) to top (3' end) to determine sequence

Chromatography

General Principle

Separates molecules in a mixture based on their interaction with two phases:

• Stationary phase (typically polar)

• Mobile phase (typically non-polar)

In reverse-phase chromatography, the polarity is swapped.

Types of Chromatography 1

Liquid Chromatography

• Stationary: Silica (polar)

• Mobile: Non-polar solvent (e.g., toluene)

HPLC (High-Performance Liquid Chromatography)

Feature	Description
Key difference	Uses high pressure and finely-ground stationary phase
Advantage	Higher resolving power (better separation)
Detection	Based on absorbance and elution time
Normal phase	Stationary = polar, Mobile = nonpolar
Reverse phase	Stationary = nonpolar, Mobile = polar

Gas Chromatography (GC)

Feature	Description
Mobile phase	Inert gas (helium, nitrogen)
Stationary phase	Thin liquid/polymer layer coating a tube
Sample requirement	Must be vaporizable
Separation basis	Polarity (polar = slower elution, higher retention time)

Gel-Filtration (Size-Exclusion) Chromatography

Molecule Size	Behavior	Elution
Molecule size: Large	Cannot enter porous beads	Elutes first
Molecule size: Small	Enters pores in beads	Elutes later (longer path)

Memory trick: Large = Left out of beads = Leaves first

Ion-Exchange Chromatography

Type	Bead Charge	Attracts	Elutes First
Cation-exchange	Bead Charge: Negative (-)	Attracts: Positive proteins	Elutes First: Negative proteins
Anion-exchange	Bead Charge: Positive (+)	Attracts: Negative proteins	Elutes First: Positive proteins

Memory aid: Cation = + charge. Cation-exchange = beads are negative to attract cations. Anion-exchange = beads are positive to attract anions.

Affinity Chromatography

• Beads are coated with a specific ligand

• Proteins with high affinity bind to beads

• Low affinity proteins elute first

• Bound proteins are released by adding free ligand (competition)

Thin-Layer Chromatography (TLC)

Component	Detail
Stationary phase	Silica gel on a plate (polar)
Mobile phase	Non-polar solvent
Process	Solvent travels up by capillary action
Visualization	UV light
Measurement	Rf value = distance spot traveled ÷ distance solvent traveled

Distillation

Purpose

Separate molecules based on boiling point differences

Types

Type	When to Use
Simple Distillation	Boiling points differ by ≥25°C
Fractional Distillation	Boiling points differ by <25°C
Vacuum Distillation	Boiling points are so high that compounds might decompose

Key insight: Vacuum lowers the boiling point, allowing separation at lower, safer temperatures.

Polymerase Chain Reaction (PCR)

Purpose

Amplify (make millions of copies of) a specific DNA sequence

Components Needed

• DNA template

• Primers (complementary to target)

• dNTPs (building blocks)

• Taq DNA polymerase (heat-stable)

• Buffer solution

PCR Cycle (Repeated ~30-40 times)

Step	Temperature	Time	What Happens
1. Denaturation	95°C	~15 sec	Double-stranded DNA separates
2. Annealing	54°C	~15-30 sec	Primers bind to single-stranded DNA
3. Extension	72°C	~1 min/kb	Taq polymerase synthesizes new complementary strands

Why Taq Polymerase?

• Derived from Thermus aquaticus (heat-loving bacteria)

• Remains active at high temperatures needed for denaturation

• Optimal temperature: ~72°C

Quick Comparison Chart

Technique vs What it Seperates/ Detects vs Basis of Seperation

Technique	What it Separates/Detects	Basis of Separation
Native-PAGE	Proteins	Size (native state)
SDS-PAGE	Proteins	Mass only
Isoelectric Focusing	Proteins	Isoelectric point (pI)
Southern Blot	DNA	Size + sequence
Northern Blot	RNA	Size + sequence
Western Blot	Proteins	Size + antibody binding
Sanger Sequencing	DNA	Termination at specific bases
Ion-Exchange	Proteins	Net charge
Size-Exclusion	Molecules	Size
Affinity Chromatography	Proteins	Ligand binding
GC/HPLC/TLC	Various molecules	Polarity
Distillation	Liquids	Boiling point
PCR	DNA amplification	N/A (copies DNA)

NMR Spectroscopy (Nuclear Magnetic Resonance)

Basic Principle

NMR uses magnetic fields to analyze the environment of specific atomic nuclei (¹H or ¹³C) within a molecule, revealing structural information.

¹H-NMR (Proton NMR)

Three Key Pieces of Information from Every Peak:

Feature	What It Tells You
Chemical shift (x-axis, δ ppm)	Electronic environment (deshielding)
Integration (area under peak)	Number of equivalent hydrogens
Splitting pattern (number of sub-peaks)	Number of neighboring hydrogens (n+1 rule)

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Chemical Shift Regions (¹H-NMR)

δ (ppm) Range	Functional Group
0 - 5	Alkanes (C-H bonds)
3 - 5	Alkanes with heteroatom nearby (O, N, halogen)
5 - 7	Alkenes (C=C-H)
6 - 8	Aromatic ring hydrogens
9 - 10	Aldehydes (R-CHO)
10 - 13	Carboxylic acids (R-COOH)

Key Concept: Higher chemical shift = more deshielding = electrons pulled away by nearby electronegative atoms or pi bonds.

Splitting Patterns (n+1 Rule)

Pattern	Number of Peaks	Neighboring Hydrogens
Singlet	1	0
Doublet	2	1
Triplet	3	2
Quartet	4	3
Quintet	5	4
Sextet	6	5
Septet	7	6
Multiplet	8+	7+

Memory aid: Neighbors + 1 = number of peaks. Hydrogens on adjacent carbons (≤3 bonds away) cause splitting.

Integration: The area under each peak is proportional to how many equivalent hydrogens that signal represents. For example, a peak integrating to 3H represents a -CH₃ group.

¹³C-NMR (Carbon NMR)

Key Differences from ¹H-NMR:

• No integration values

• No splitting patterns

• Just focuses on chemical shift (number of unique carbon environments)

Chemical Shift Regions (¹³C-NMR)

δ (ppm) Range	Functional Group
0 - 70	Alkanes (sp³ carbons)
90 - 120	Alkenes (sp² carbons)
110 - 160	Aromatic ring carbons
160 - 200	Carbonyls (C=O)

IR Spectroscopy (Infrared)

Purpose

Identifies functional groups based on bond vibrations. Only bonds with a dipole momentabsorb IR radiation.

Axes

• X-axis: Wavenumbers (cm⁻¹) — reciprocal centimeters

• Y-axis: Percent absorbance (or transmittance)

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Important IR Absorption Regions

Wavenumber (cm⁻¹)	Functional Group	Peak Shape
1700 - 1750	Carbonyls (C=O)	Sharp
1720 - 1740	Aldehydes	Sharp
1700 - 1725	Ketones	Sharp
1735 - 1750	Esters	Sharp
1700 - 1725	Carboxylic acids	Sharp
3200 - 3600	O-H groups (alcohols, acids)	Broad
3300 - 3400	Amines (N-H)	Sharp
	1° Amine → 2 peaks
	2° Amine → 1 peak

Quick Recognition Tips IR

What You See	What It Means
Sharp peak ~1700	Carbonyl group present
Broad peak ~3400	O-H (alcohol or carboxylic acid)
1-2 peaks ~3300	Amine (1° = two peaks, 2° = one peak)
No peaks	Molecule may be symmetric (no dipole)

UV-Vis Spectroscopy

Principle

• Measures absorption of ultraviolet/visible light

• Involves electron transitions between HOMO and LUMO (highest occupied / lowest unoccupied molecular orbitals)

Conjugation Rule

More Conjugated Pi Bonds	Effect
More double bonds in conjugation	Smaller HOMO-LUMO gap
Smaller gap	Lower energy light absorbed
Lower energy	Longer wavelength absorbed

Color and Absorption

Complementary color rule: If a molecule absorbs green light, it appears red (the complementary color).

Autoradiography

Purpose

Visualize the location of radioactive substances in a molecule or structure.

How It Works

1. Radiolabeled sample is placed against photographic emulsion containing silver halide crystals

2. Radiation from the sample converts silver halide → metallic silver

3. Produces a visible image showing where radioactivity is located

MCAT Context

Used to detect radiolabeled probes in:

• Southern blots (DNA)

• Northern blots (RNA)

• Western blots (Protein detection via radiolabeled antibodies)

X-Ray Crystallography

Purpose

Determine the 3D structure of molecules, especially proteins.

How It Works

1. Molecule is crystallized

2. X-ray beam is directed at the crystal

3. X-rays diffract (scatter) in specific patterns

4. Angles and intensities of diffracted rays are measured

5. Computer reconstructs 3D electron density map → 3D structure

Key point: Requires crystallization of the molecule, which can be challenging for proteins.

Immunoprecipitation

Purpose

Purify a specific protein from a solution.

How It Works

1. Antibody-coated beads (specific to target protein) are added to solution

2. Antibodies bind to the protein of interest

3. Bead-protein complexes are isolated by:

   • Magnetic extraction (if magnetic beads)

   • Centrifugation (pellet formation)

Key point: Uses antibody specificity to "pull down" one protein from a complex mixture.

Radioimmunoassay (RIA)

Purpose

Determine the concentration of a specific protein in a sample.

How It Works (Competitive Binding Assay)

Step	Action
1	Plate wells are coated with primary antibody specific to target protein
2	Radiolabeled protein (tagged with ¹²⁵I) is added → binds antibody
3	Measure initial radiation (gamma counting) → gives baseline
4	Add unknown sample containing target protein
5	Unknown protein competes with radiolabeled protein for antibody binding sites
6	Some radiolabeled protein is displaced
7	Measure final radiation
8	Difference in radiation = concentration of protein in unknown sample

Key Concept

• More protein in unknown sample → more displacement of radiolabeled protein → lower final radiation count

• This is a competitive assay (similar to competitive ELISA)

Mass Spectrometry (Mass Spec)

Purpose

Determine molecular weight and help identify molecular structure.

Process

1. Sample is vaporized and ionized

2. Molecule collides with electron → ejects electron → becomes charged radical

3. Charged radical may fragment or be detected intact

Axes

• X-axis: Mass-to-charge ratio (m/z) — essentially the mass using most abundant isotopes (¹²C, ¹H, ³⁵Cl)

• Y-axis: Relative abundance/intensity (%)

Key Peaks to Identify

Peak	What It Represents
Base peak	Tallest peak; most abundant fragment (NOT always the intact molecule)
Molecular ion peak (M)	The intact molecule; its m/z = molecular weight
M+1 peak	Due to ¹³C isotope (1.1% natural abundance per carbon)
M+2 peak	Due to ³⁷Cl or ⁸¹Br isotopes

How to Use M+1 and M+2 Peaks

M+1 Peak → Count Carbons

• ¹³C is 1.1% abundant

• Formula: %M+1 ÷ 1.1 ≈ number of carbons

• Example: M+1 = 4.4% → 4.4/1.1 = 4 carbons

M+2 Peak → Identify Cl or Br

Halogen	Isotope Pattern	Ratio (M : M+2)
Chlorine (Cl)	³⁵Cl : ³⁷Cl	3:1
Bromine (Br)	⁷⁹Br : ⁸¹Br	1:1

Example interpretations:

• M peak = 90%, M+2 = 30% → 90:30 = 3:1 → Chlorine present

• M peak = 90%, M+2 = 90% → 90:90 = 1:1 → Bromine present

ELISA (Enzyme-Linked Immunosorbent Assay)

Purpose

Determine the concentration of a specific molecule (antigen) in a sample.

Key Components

Component	Role
Primary antibody	Binds specifically to the target molecule
Secondary antibody	Binds to primary antibody; linked to an enzyme(often HRP = horseradish peroxidase)
Enzyme + substrate	HRP reacts with oxidizing agent (e.g., H₂O₂) → color change
Spectrophotometry	Measures absorbance → determine concentration
Standard curve	Serial dilutions of known concentration used for comparison

Indirect ELISA

Detects: Antibodies in a sample (or antigen presence)

Step	Action
1	Coat wells with the antigen of interest
2	Add primary antibody (from patient sample) → binds antigen
3	Wash to remove unbound antibodies
4	Add secondary antibody (enzyme-linked, anti-human) → binds primary antibody
5	Wash to remove unbound secondary antibody
6	Add substrate → enzyme catalyzes color change
7	Measure absorbance → compare to standard curve

Key point: The antigen is fixed to the plate FIRST.

Sandwich ELISA

Detects: Antigen in a sample

Step	Action
1	Coat wells with capture antibody (primary antibody) specific to target
2	Add sample containing antigen → binds to capture antibody
3	Wash to remove unbound antigen
4	Add detection antibody (secondary antibody, enzyme-linked) → binds antigen
5	Wash to remove unbound detection antibody
6	Add substrate → enzyme catalyzes color change
7	Measure absorbance → compare to standard curve

Key point: The antigen is "sandwiched" between two antibodies. The capture antibody is fixed to the plate FIRST.

Indirect vs. Sandwich ELISA Comparison

Feature	Indirect ELISA	Sandwich ELISA
What's coated on plate first	Antigen	Capture antibody
What is detected	Antibodies (or antigen)	Antigen
Number of antibodies binding antigen	One (primary)	Two (capture + detection)
Specificity	Moderate	High (two antibodies recognize antigen)

Memory aid: Sandwich = "sandwiched" between two antibodies like bread

Edman Degradation

Purpose

Sequence the amino acids of a protein from the N-terminus, one residue at a time.

How It Works

Step	Action
1	Phenyl isothiocyanate (PITC) added → reacts with N-terminal amino acid
2	N-terminal residue cyclizes and cleaves off from the rest of the polypeptide
3	The rest of the polypeptide remains intact (shortened by one residue)
4	The cleaved PTH-amino acid is analyzed by chromatography
5	Repeat for each subsequent amino acid

Limitations

• Only accurate for polypeptides less than 50 residues

• Cannot sequence longer proteins in one run

Key concept: Sequential removal from N-terminus without destroying the rest of the chain.

Gram Staining

Purpose

Differentiate bacteria into Gram-positive or Gram-negative based on cell wall structure.

Procedure

Step	Reagent	Action
1	Crystal violet	Primary stain (purple); enters all bacteria
2	Iodide	Mordant; binds crystal violet, traps it in cell wall
3	Alcohol	Decolorizer; removes dye from Gram-negative bacteria
4	Safranin	Counterstain (pink); stains decolorized bacteria

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Results

Type	Color	Cell Wall Structure
Gram-positive	Purple	Thick peptidoglycan layer; retains crystal violet during alcohol wash
Gram-negative	Pink	Thin peptidoglycan layer sandwiched between two lipid bilayers; loses crystal violet, takes up safranin

Key distinction: Peptidoglycan thickness determines whether the purple dye is retained during the alcohol wash.

RFLP (Restriction Fragment Length Polymorphism)

Purpose

Identify differences between homologous DNA sequences (e.g., wild type vs. mutant) based on fragment lengths after restriction enzyme digestion.

Key Concepts

Term	Definition
Restriction enzyme	Enzyme that cuts DNA at specific palindromic sequences (same sequence read 5'→3' on both strands)
Polymorphism	A difference in DNA sequence between individuals/alleles

Procedure

Step	Action
1	DNA samples (WT and mutant) are digested with restriction enzymes
2	If mutation creates or destroys a restriction site → different fragment lengths
3	Fragments separated by gel electrophoresis
4	Compare band patterns between samples

Interpretation

• Same pattern = no difference in restriction sites (no polymorphism detected)

• Different pattern = mutation altered a restriction site, changing fragment lengths

Application: Genetic testing, forensics, paternity testing

Salting Out and Dialysis

Purpose

Purify proteins from a solution.

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Salting Out (Protein Precipitation)

Concept	Explanation
Mechanism	Salt ions compete with proteins for water of solvation
Effect	At high salt concentration, proteins lose water shell → aggregate and precipitate
Selectivity	Different proteins precipitate at different salt concentrations

Common salt used: Ammonium sulfate ((NH₄)₂SO₄)

Key concept: You can selectively precipitate your protein of interest by adding just enough salt.

Dialysis (Salt Removal)

Step	Action
1	Place precipitated protein solution in dialysis bag(semipermeable membrane)
2	Submerge bag in hypotonic solution (low/no salt)
3	Small ions (salt) diffuse out of bag through pores
4	Large proteins cannot pass through membrane → remain in bag
5	Eventually, salt is removed and protein is purified

Key concept: Semipermeable membrane allows small molecules through but retains large proteins.

Reducing Sugar Tests

What is a Reducing Sugar?

A sugar that can act as a reducing agent because it has a free aldehyde or free ketone group (open-chain form).

Key Rules

• All monosaccharides are reducing sugars (can undergo mutarotation → open-chain form)

• Some disaccharides are reducing (e.g., maltose); others are NOT (e.g., sucrose)

• Sucrose is non-reducing because the glycosidic bond is 1→2 (links both anomeric carbons), preventing mutarotation

• Maltose is reducing because one anomeric carbon is free

Three Reducing Sugar Tests

Test	Reagent	Detects	Positive Result
Tollen's Test	Reagent: [Ag(NH₃)₂]NO₃ (silver-ammonia complex)	Detects: Aldehydes	Positive: Silver mirror (elemental Ag precipitates)
Benedict's Test	Reagent: Na₂CO₃ + Na citrate + CuSO₄	Detects: Aldehydes	Positive: Brick-red precipitate (clear blue → red)
Fehling's Test	Reagent: Fehling's A: CuSO₄ + Fehling's B: KNa tartrate + NaOH	Detects: Aldehydes	Positive: Brick-red precipitate (clear blue → red)

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Important Notes for All Three Tests

• Ketones do NOT react (negative test) UNLESS they are α-hydroxy-ketones

• All three detect free aldehyde groups

• Positive result confirms presence of a reducing sugar with a free aldehyde

cDNA Libraries

Purpose

Create and store complementary DNA (cDNA) from eukaryotic mRNA so proteins can be expressed in bacterial vectors.

Why cDNA Instead of Genomic DNA? Problem vs Solution

Problem	Solution
Eukaryotic genes contain introns	cDNA is made from mRNA (already spliced)
Prokaryotes cannot remove introns	cDNA has no introns → can be expressed in bacteria

Example application: Producing human insulin in E. coli

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Procedure

Step	Action	Key Player
1	Isolate mRNA for protein of interest	mRNA has poly-A tail
2	Add oligo-dT primer (thymine repeats with free 3'-OH)	Anneals to poly-A tail
3	Add reverse transcriptase + dNTPs	Synthesizes cDNA strand → forms cDNA-mRNA hybrid
4	Hydrolyze mRNA with alkaline solution	Leaves single-stranded cDNA
5	Add DNA polymerase + primer	Synthesizes complementary strand → double-stranded cDNA
6	Insert ds-cDNA into bacterial plasmid using restriction enzymes	Cut and ligate into vector
7	Transform bacteria → bacteria produce protein of interest	Expression system