Overview of 2DE

Complex mixture of proteins

Separate proteins by charge in first dimension (IEF)

Separate proteins by size in second dimension (SDS-PAGE)

Denature and solubilize in solution (Sample prep)

Individual proteins isolated as distinct protein features within a gel matrix

Sample Preparation

Disrupt tissues and cell membrane Mechanical force and detergents

GSI productsProcedure

Non-ionic and zwitterionic detergents

Sample Buffer I

Remove non-protein componentsCentrifugation and degrading enzymes

Sample Buffer IIRNAse and DNAse

Disrupt complexes and ‘linearize’ proteinschaotropic agents (Urea)

reducing agents (DTT)detergents

Rehydration bufferIPG loading bufferSample buffer III

Eliminate highly abundant proteins Cibachron dye mini-columns

Remove salt from fractionated or affinity purified samples Dialysis kits and buffers

Isoelectric Focusing- Separation by Charge

-CathodeAnode

+

Acidic Basic pH 3 4 5 6 7 8 9 10

Proteins are amphoteric (contain acidic and basic residues)

For every protein there is a pH at which its net charge is 0. This is its isoelectric point (pI)

Above its pI, a protein has an overall negative charge and will migrate toward the positively charged anode

Below its pI, a protein has an overall positive charge and will migratetoward the negatively charged cathode

At its pI, a protein does not move (focuses into a single band)

Final Result of IEF

Acidic Basic

Proteins focused into distinct bands

BPB

Methods for isoelectric focusing

Carrier-ampholyte tube gels

pH gradient created by discontinuous buffering system and carrier ampholytes

Immobilized pH Gradient(IPG) strips

pH gradient fixed in gel by covalently linking amphoytesto acrylamide when gel gradient is poured.

Investigator Tube Gel Apparatus

Capacity: 15 analyticalor 8 preparative tubes

Investigator IPG pHaser

Investigator IPG pHaser

Runs up to 10 IPG strips

Compatible with all brands of IPG strips

Tube gels vs IPG strips

Benefits of tube gels

No rehydration step- saves one day.May be better for some proteins-membrane, hydrophobic.

Benefits of IPG strips

Immobilized pH gradient eliminates cathodic and anodic drift.Higher volume of sample can be loaded.Less likely to become damaged in 2DE procedure.Less labor involved.

Second dimension SDS Page- Separationof proteins by mass

Anode +

Coat focused proteins with SDSGSI products: Equilibration buffers I and II

Place strip/tube directly onto 2nd D gel

GSI products: 2-D Running System-tank, power supply, chiller,precast slab gels, gel casting reagents, premixed buffers

Negatively charged proteinsmigrate toward + anode

Typical results following 2DE

AcidicProteins

BasicProteins

High MW

Low MW

Investigator 2DE Electrophoresis System

Peltier chiller

Programmable Power Suppy

Capacity: 5 singleor 10 double gels

Single power supplyruns tube gels, IPGstrips and slab gels

Detection of protein features

Staining method Detection limits

300ng

30ng

5ng

5ng

1ng

Standard Coomassie

Colloidal Coomassie

Fluorescent Dyes

Non-destructive Silver

Destructive Silver

Standard Coomassie Staining

General method

Add stain with fixative and incubate 4hrs to overnight.Destain in 40% Methanol, 10% acetic acid.

Benefits Easy and consistentMass-spec friendly

DisadvantagesLeast sensitive stainRequires long incubation times and destaining

Colloidal Coomassie Staining

General MethodIncubate gel in colloidal solution for minutes or hoursDestain with water (if required).

BenefitsMass spec friendlyVery fastEnvironmentally friendly/ less hazardousMore sensitive than standard coomassieNo special visualization requirements

DisadvantagesDetection limit 10-50 times lower than fluorescent dyesor silver.

Staining using Fluorescent dyes

General methodIncubate in dye for 1 hour to overnightDestain (if desired)

BenefitsFast and easy to useNon-toxicVery sensitiveLinear over a broad range (ng to mg)Mass spec friendly

DisadvantagesRequires UV source for visualization

BenefitsMost sensitive stain (when gluteraldehyde is used)

DisadvantagesLong and tedious procedureLabor intensiveHazardousVery sensitive to wash and development timesLinear over a very narrow rangeProtein specific staining (some do not stain or stain negatively)Most sensitive method (destructive) not compatiblewith mass spec

Pros and Cons of Silver Staining

Examples:

If the sensitivity for Coomassie equals 40-50ng per feature, then 1,000 features could be detected starting with 50-100ugs sample.To visualize all the features in a sample containing 10,000 features you need to start with 500ug-1mg total protein.

If the sensitivity for silver equals 1-5ng per feature, then 1000 features could be detected starting with 1-5ug sample.To visualize all the features in a sample containing 10000 features you need to start with 10-50ug total protein.

Note: These examples assume that all proteins in the sampleare present in equal amounts, not the case in real life.

How much sample should I load?

The amount of sample required depends on boththe staining method, AND the complexity of the sample

Finding what you’re looking for

2DE of crude fractions using broad range IEF gels can only provide information on relatively abundant proteins (high copy number). Ifyou want to detect moderate to rare proteins you must reduce the complexity of the sample or limit the scope of the search.

The ‘simple’ eukaryote, yeast, has approximately 6000 genes but can produce over 12,000 protein features due to post-transcriptional and post translational modifications. To ‘see’ a rare protein within a crude extract, 20-2000mgs of totalprotein would need to be loaded onto the 1st dimension gel.

Therefore

Methods for finding the ‘interesting’ proteins

Prefractionate- Reduce the complexity of each sample by fractionating into nuclear, cytoplasmic, mitochondial, microsomal or other distinct compartments.

Remove highly abundant proteins

Purify complexes- Enrich for specific activities, or complexcomponents.

Use narrow range IEF gels- Increase the amount of sampleyou can load on a gel while increasing resolution within narrow PI ranges (3-6, 5-7,6-8,7-9). The use of zoom gels (onepoint pH spread) allows loading of up to 40mgs starting sample.

2-D gel of sample before (left) and after (right) treatment with albumin depletion kit.