Electrophoresis and 2D Gel Analysis

What is Electrophoresis ?The migration of a charged particle (protein) through a separation matrix toward the opposite charged electrode

Cathode Anode

Anion

Cation

Types of Electrophoresis1D Gel Electrophoresis- DNA, RNA, and Protein

Agarose –low resolution, general use for RNA and DNAPolyacrylamide – High resolution

Size determination, DNA Sequencing, gel shift, etcSDS-PAGE-ProteinPulse Field- DNA separation of very large fragments of genomic DNA

2D Gel ElectrophoresisIsoelectric focus and PAGE –ProteinDIGE-Differential gel electrophoresis

Two samples labeled with different fluorophores are mixed and run on a single 2d gel

Capillary Electrophoresis (CE)Linearized polyacrylamide, PVPAutomated DNA Sequencing, genotyping

Micro-channel/ Micro-fluidic ElectrophoresisLab-on-a-Chip

Electrophoresis and Biomolecules

DNA and RNA:Phosphate backbone is negatively charged (anionic) and moves toward positive charge (anode)

Separated based on size and sometimes structure

faster migration

slower migration

(-)

(-)

(-)

(-)

(-)

Proteins:Proteins can have both positive and negative charges Proteins can be acidics or basic [amphoteric]

In their native state, the overall charge is variable

Electrophoresis and Biomolecules

Anionic detergents [SDS] denature secondary and non–disulfide–linked tertiary structures and impart a negative charge in proportion to its mass.

SDS is therefore required when running standard gels to determine size in KDa

R-Groups and Interactions

Electrophoresis and Biomolecules

Isoelectric Point (IEP)- also known as pI pH at which a protein has a neutral charge

loss or gain of protons H+ in a pH gradientThat is to say…

In a pH below their pI, proteins carry a net positive charge and for in a pH above their pI, they

carry a net negative charge.

Proteins:

Isoelectric Focusing (IEF):

“First Dimension” in 2-D gel

Utilizes a special pH gradient strip and high voltage electrophoretic equipment

Variable pH Ampholytes embedded in Acrylamide

Proteins separate of according to their isoelectric point (pI)

The equilibrium that a protein establishes at a specific pH on the IPG is an electro-chemical “focusing”.

When a protein moves away from its IEF pH, its charge will deviate from a neutral state and either gain charge or lose charge and finding its way back to the “comfortable” state of neutral.

Proteins:

IEF apparatus from Bio-Rad

1D Gel Electrophoresis

1D Gel Electrophoresis-SDS PAGE

Standard use for protein separation based on size (MW)

Uses sodium dodecyl sulfate to denature protein and provide negative charge for mobility

Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio and structure

Degree of resolution determined by % acrylamide

SDS PAGE Principles

CATHODE

ANODE

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+++++++

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_ _ _ _ _ _ _

_

_ __ _

_ _ _ _

_ _ _

_ _ _ _ _

SO Na4+

Sodium Dodecyl Sulfate

_

Components of SDS PAGE Gel

B-Mercaptoethanol /DTT [a reducer]

Added to prevent oxidation of cysteines and to break up disulfide bonds within the proteins

Sodium dodecyl sulfate [negatively-charged ionic detergent]

binds to the vast majority of proteins at a constant ratio of 1.4 gm SDS/gm protein and provide a constant charge to mass ratio for electro-mobility

GlycerolProvides density for gel loading

Bromophenyl blue A tracking dye used for visualization purposes during a run

Stacking GelIs prepared w/Tris/HCL buffer pH 6.8, ~2pH units lower than running buffer. Large pore polyacrylamide used to align and create a thin starting zone of the protein of apx. 19um on top of the resolving gel. Resolving GelSmall pore polyacrylamide gel (3 - 30% acrylamide monomer) typically made using a pH 8.8 Tris/HCl buffer. Resolves protein ~24 – 205 kDa

Running BufferTris/Glycine: Glycine(pKa=9.69) is a trailing ion (or slow ion). In other words it runs through the gel slower then the slowest protein at a pH above 8.0.

Components of SDS PAGE Gel

Polyacrylamide Chemistry

N,N'-methylene-bis-acrylamide

Ammonium persulfate

{Free radical generator}

Tetramethylethylene diamine [TEMED]

Catalyst {O2 Scavenger}

Polyacrylamide

Acrylamide monomer

TEM imagePore size of gel is determined by total amount of acrylamide and bis-acrylamide

Affect of % Acrylamide

200

200200

200

45

45

4545

45

6.5

200

Staining Polyacrylamide Gels Coomassie Blue Stain- can usually detect a 10-50 ng protein per bandBlue Safe Stains -Similar to Coomassie. Destaining optional or water rinse. (8

ng/band)BioSafe BlueSimplyBlueGelCodeInstantBlue-destaining not recommended

Silver Staining- 50 times more sensitive than Coomassie Blue. (0.3ng/BAND) Fixation [Acetic acid-methanol]Sensitize gel with sodium thiosulfate Stain with silver solutionRinse with waterDevelop with formaldehyde and carbonate followed by stopping with Glacial acetic

Fluoresecnt Stains –almost as sensitive as Silver but requires excitation sourceFlamingo Fluorescent Gel Stain Deep Purple* Total Protein Stain SYPRO* Ruby Protein Gel Stain Krypton Protein StainIR stains

Coomassie –Protein Binding Sulfonic acid group interacts with positively charged amine R

groups

Basic amino acids including arginine, lysine and histidine but weakly with histidine, tyrosine, tryptophan and phenylalanine

Interactions in its anionic form [-]

ElectrostaticIonicVan der Waals

Hydrophobic

Destaining GelsMost gels require destaining to see banding and to eliminate background stain

for high resolution.

Gels with abundant protein need not be destained when using certain SafeBlue stains such as InstantBlue

Coomassie blue destainingUsually requires acetic acid , methanol, and water

Safe Blue destainingUsually requires water rinse

Silver StainsSome methods use Potassium Ferricynide -Sodium Thiosulfate solutionsSome methods use Sodium chloride -Cupric sulfate -Sodium thiosulfate

pentahydrate.

Some destaining may require a stop solution including 10% Acetic acid

2D Gel Electrophoresis

2D Gel Electrophoresis

Yeast Proteome: 50 ug protein loaded, pH 4-8 ampholines, 10% slab gel, silver stain.  

2D Gel Electrophoresis

Separation of hundredsof proteins based on

-pI-MW

Up to 10,000 proteins can be seen using optimized protocols

Why 2D Gels

Oldest method for large scale protein separation (since 1975)

Popular method for protein display and proteomics-one spot at a time

Can be used in conjunction with Mass Spec

Permits simultaneous detection, display, purification, identification, quantification, pI, and MW.

Robust, reproducible, simple, cost effective, scalable

Provides differential quantification using Differential 2D Gel Electrophoresis (DIGE)

Processes involved in 2D gel electrophoresisProtein isolation and quantification

Isoelectric focusing (first dimension)

SDS-PAGE (second dimension)

Visualization of proteins spots with Dye

Identification of protein spots with Mass Spec

Bioinformatics

Sample Preparation• Sample preparation is key to successful 2D gel experiments

• Must select appropriate method to get selected proteins from cellular compartment of interest

• Membrane proteins, nuclear proteins, and mitochodrial proteins require special steps

• Must break all non-covalent protein-protein, protein-DNA, protein-lipid interactions, disrupt S-S bonds

• Must prevent proteolysis, accidental phosphorylation, oxidation, cleavage, ect..

• Must remove substances that might interfere with separation process such as salts, polar detergents (SDS), lipids, polysaccharides, nucleic acids

• Must try to keep proteins soluble during both phases of electrophoresis process

• Must quantify protein

Protein Solubilization2-20 mM Tris base (Carrier ampholytic buffer)5-20 mM DTT (to reduce disulfide bonds)8 M Urea (neutral chaotrope)

Increases the solubility of some proteins

Chaotropic agents interfere with stabilizing non-covalent forces (hydrogen bonds, van der Waals forces, and hydrophobic)

4% CHAPS Detergent (3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)

pH of 5-7

Zwitterionic detergent (electronically neutral-has a both Neg and Pos useful for varible charged peptides )

Protects the native state of proteins

Better when downstream apps include IEF because no affect on pH gradients

IEF and IPG (immobilized pH Gradient)

Strip of paper Made by covalently integrating acrylamide and variable pH ampholytes

Separation on basis of pI, not MW

Available in different pH ranges

3-104-85-7

Requires very high voltages (5000V)and long period of time (10h)

pH 3 4 5 6 7 8 9 10

I IPG Strips Contain Ampholytes

Ampholytes are molecules that contain both acidic and basic groupsProtein will migrate in the Matrix and will find their pH equilibrium (pI)

The Second Dimension …Running the Gel

SDS GelIPG strip-pressed down into the SDS-PAGE gel

Positive electrode

Negative electrode

Similar mw but different pI

Similar pI but different mw

pH 3 4 5 6 7 8 9 10

Different IPG pH ranges yield Different Results

pH 4 pH 5

pH 5 pH 7

pH 4 pH 9

Gel Stains - Summary

Stain Sensitivity (ng/spot) Advantages

Coomassie-type 5-10 Simple, fast

Silver stain 1-4 Very sensitive, laborious

Copper stain 5-15 Reversible, 1 reagentnegative stain

Zinc stain 5-15 Reversible, simple, fasthigh contrast neg. stain

SYPRO ruby 1-10 Very sensitive, fluorescent

2D Gel Results

• 401 spots (peptides or PTM) identified

• 279 gene products

2D Gel Post Analysis

Compare gel images and determine what bands/spots are different

Requires software to compare gels

Apparent difference- Need to extract spot for MS

Trypsin Digestion of Gel spot

Cut out spot

Extracting a Gel Spot Run Mass spec

Differential 2D Gel Electrophoresis [DIGE]Allows you to mix samples and run a single 2d gel for

comparative and quantitative purposes

Fluorescent stain

Cy3-- Normal liverCy5--TumorBoth

Conclusions• 2D gel electrophoresis is a popular method for protein display,

separation, visualization, and quantitation

• A good precursor to MS, but not required

• 2D gels provide pI, MW data, and photodocumentation

• Web tools are now available that permit partial analysis and comparison of 2D gels using software and simulators

• 2D gels are fun to run