2-Dimensional Electrophoresis

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Proteomics Pathway

Proteomics Pathway

History of 2-DE

n1956

-paper & starch gel

n1975

-Coupling of IEF & SDS-PAGE

n IPG-IEF

uImmobilized pH Gradient

uIsoElectric Focusing

uCan use pH gradients 3-12

2DE-Techniques

History of IEFn NEPHGE (Non-Equilibrium pH

Gradient Electrophoresis)

n IPG IEF (Immobilized pH Gradient IEF)

IEF – pH Rangen Range of pH 3-12

n Majority falling between pH 4-7

n Ex: BIO-RAD commercial strip pH range

IEF

1.Rehydration

2.Wick

3.Conditioning time

4.Voltage ramping

5.Final focusing

6.Hold step

2DE-Techniques

n SDS-PAGE

uSodium Dodecyl Sulfate

u PolyAcrylamide Gel Electrophoresis

u Able to

FDisrupt most non-covalent protein interactions

FSolubilize membrane proteins

2-Dimensional Electrophoresis 2-Dimensional Electrophoresis

1D (IEF) Equilibration

2D (SDS-PAGE)Identification

SDS-PAGE

IEF

+

Sample preparation

n Solubility, size, charge, isoelectric point

n No universal method due to the diverse nature of samples.

n Essential to minimize protein modifications.

n Protease

n Salt

Cell / Tissue Preparationn Break up the tissue while it is still frozen or at

liquid nitrogen (Pestle, Mortar, Homogenizer)

n In cells, washed in phosphate-buffered saline (PBS) and solubilized in sample buffer

n Nucleic Acid Removal :

u Ultracentrifugation of Complexes w/ ampholytes

u Addition of suitable (Protein-free) endonucleases

n Sample fractionation (sub-cellular, HPLC, adsorption chromatography, selective precipitation, increased solubility extraction)

Troubleshooting

Application of Proteomics

n Introductionn Organic dyes and silver stainsn Reverse stainsn Colloidal dispersion stainsn Organic fluorophore stainsn Metal chelate stains

Detecting proteins in polyacrylamide gels and on electroblot membrane

Introduction

n History of stains used to visualize proteinsu Bromophenol blue Amido black

Coomassie blue R-250 Coomassie blue G-250 Silver staining Colloidal staining Negative staining SYPRO Red, Orange, Ruby staining

Organic dyes

n Organic dyesu Coomassie blue: wide spread popularity

since its introduction in the early 1960s, capable of detecting as little as 30-100ng (limit 8-10ng)

u Amido black : largely relegated to medium sensitivity, colorimetric detection of electro blotted proteins on PVDF and nitrocellulose membranes

Coomassie blue

Silver Stainsn Silver staining

u Detection sensitivity: 0.6-1.2ng

u Alkaline/silver diamine and acidic/silver nitrate are most commonly used

F Alkaline/silver diamine: histological producers and use ammonium hydroxide to form soluble silver-diamine complex

F Acidic/silver nitrate: photographic procedures and rely upon gel impregnation with silver ions at acidic pH

u Cons: Some aa-modification

Silver Staining

Reverse stainsn Reverse stains were developed specifically to

improve protein recovery from polyacrylamide gels

n Staining is quite rapid, usually requiring 5-15min to complete and the biological activity of proteins is often preserved

Copper staining

Zinc staining

Limit Detection: 10-20 ng per band Compatible w/ MALDI-TOF

Colloidal dispersion stains

n The stains contain fine particles with high affinity for protein

n The stains are not generally used for detection of proteins in polyacryl-amide gels (PAGE)

n India ink stain: simple to perform and inexpensive

n Colloidal metal stains: gold staining

Organic fluorophore stainsn Fluorescent detection of proteins after

electrophoresis is gaining popularity with laboratories engaging in large-scale proteome research

u Covalently bound fluorophores: more recently propyl-Cy3 and methyl-Cy5 dyes have been utilized

u Noncovalently bound fluorophores (2-10 ng):

F SYPRO OrangeF SYPRO Red

SYPRO Orange staining

SYPRO Red staining

Metal chelate stainign The metal chelate are a relatively new family of

protein visualization reagents developed to be compatible with modern proteomics research

u Colorimetric metal chelate stains: disulfonate/ferrous(600ng), copper phthalocyanine tetrasulfonate(10-20ng)

u Luminescent metal chelate stains: SYPRO Ruby 2-DE and IEF stains allow one-step, low

background staining of proteins in polyacrylamide gels without resorting to lengthy destaing steps (0.25-1 protein/mm2).

SYPRO Ruby staining

Properties of an ideal general protein stain

n The seven Ss of superior stainingu Safety no hazardous materials

u Sensitivityideally subnanogram detection

u Simplicity simple incubation in 1 solution (< time)

u Specificitydetect proteins (no lipids, CHOs, NAs)

u Speed rapid w/ no destaining step

u Stability stable staining solutions at room T

u Synergy compatible w/ a wide range of applications

2-D Staining

Conclusionn With the development of proteomics into a

high-throughput approach for the study of global protein regulation, new demands are being placed on protein visualization methods

n Newer stains such as Zinc-imidazole, and the noncovalently fluorescent stains (SYPRO dyes) are available to meet the growing demand of modern protein micro-characterization technologies

Image Analysis of Two-dimensional Gels

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2DE-Image Analysis – Introduction

n Scanning of gel image

n Image processing

n Spot detection and quantitation

n Gel matching

n Data analysis

n Data presentation and interpretation

n Creation of 2-DE databases

2DE – Data acquisitionn Image acquisition devices mainly used for 2-DE

XXGel (fluorescent-labeled)

XXGel silver-, color-stained

XPhosphor image plate

XxX-ray film (radiolabeled sample)

Fluorescent

imagerStorage

phosphor imager

Densitometer White-light or

laser

Camera TV/CCD

Scanning device

Information carrier

2DE – Digital image processing

n To determining the shape and size of protein spot

u Smoothing operator � statistical noise (Gaussian function)

u Contrast enhancement- spot / background

u Edge detection

u Background subtraction

Rolling disk

BackgroundSubtraction

2DE - Protein spot detection and quantitation

n X/y position, shape parameters, integrated spot intensity

-> spot detection

n Detection algorithm:

u Gaussian fitting, Laplacian of Gaussian spot detection

u The line analysis & chain analysis

u Watershed transformation (WST)

2DE - Gel matchingn The comparative analysis of alteration on

protein spot expression

n Chemical & physical features uNo �perfect� geluNo exactly same position

n Two types of matching:uPixel LeveluSpot level

n Use characteristic spots as landmarks

2DE - Data analysisn The evaluation of quality assurance

n The determination of qualitative

u The normalization of the individual spot intensities is based in the average intensity of the related gel image.

n The determination of quantitative change in protein expression

u t-test, Mann-Whitney test, correspondence analysis

2DE - Data presentation

n Each detected protein spot should have an assigned Mr/pI.

n Graphs or bar charts can be used to compare spot quantitation and reflect alteration trends of spots.

2DE - Databasesn Construction of 2-DE database

u If a protein spot is marked on the image, the descriptive information should appear

u If the protein name or other descriptive information is known, the location of this protein on the image has to be shown and annotated

n Comparing internet-distributed 2-DE databases