2D Electrophoresis

Protein solubilization• Chaotropes ;urea - efficient in disrupting hydrogen bonds ;thiourea - breaking hydrophobic interactions • Nonionic and/or zwitterionic detergents ; NP-40, Trit

2D Electrophoresis

Electrophoresis: the movement of ions in an electric field.

Electrophoretic techniques exploit the fact that different ions have different mobilities

in an electric field and so can be separated by electrophoresis

Where, μ = free electrophoretic mobility, (μ), with units of (cm2 volt-1 sec-1)

K = a constant (embodying the Faraday constant and Avogadro's number)

q = net charge on the protein (atomic charges/protein molecule)

f = frictional coefficient

Properties of proteins

• Hydrophobicity

;Solubility, location, globularity etc.

• UV absorbity

; depends on the Tyr and Trp content

• Isoelectric point (PI)

; pH at which a molecule has no net charge.

pI of aspartate = 1/2(1.88 + 3.65) = 2.77

pK1=2.32pK2=9.60

pI of Glycine = 1/2(2.32 + 9.60) = 5.96

Enolase (NP_417259 )

5.3245650.4172

0

Isoelectric Point Molecular Weight

# Phosphates

• Isoelectric point (PI)

; pH at which a molecule has no net charge

•MW calculation: MW = ΣAAi x MWi

Residue Weight Residue Weight

2D electrophoresis

• Cell disruption

• Protein solubilization

• Prefractionation (optional)

• Isoelctric focusing (IEF)

• Equilibrium of IEF strip

• SDS PAGE

• Detection of protein spots

• Image analysis and Spot picking

• Protein spot identification

• high pressure (e.g French press)

• homogenization with glass beads and a bead beater

• a rotating blade homogenizator.

• Removal and/or inactivation of interfering compounds (protease, salt, lipid, polysaccharide, nucleic acid, etc)

No salt 30mM NaCl

Salt interference (E Coli extract)

2D electrophoresis

• Cell disruption

• Protein solubilization

• Prefractionation (optional)

• Isoelctric focusing (IEF)

• Equilibrium of IEF strip

• SDS PAGE

• Detection of protein spots

• Image analysis and Spot picking

• Protein spot identification

Protein solubilization

• Chaotropes

;urea - efficient in disrupting hydrogen bonds

;thiourea - breaking hydrophobic interactions

• Nonionic and/or zwitterionic detergents

; NP-40, Triton X-100 - not very effective in solubilizing very hydrophobic membrane proteins.

; CHAPS, sulfobetaines (e.g SB 3-10 or ASB 14) – better for hydrophobic proteins

• Reducing agents

; DTT, dithioerythritol (DTE), tributylphosphine (TBP), carboxyethyl)phosphine (TCEP)

tris(2-2D electrophoresis

• Cell disruption

• Protein solubilization

• Prefractionation (optional)

• Isoelctric focusing (IEF)

• Equilibrium of IEF strip

• SDS PAGE

• Detection of protein spots

• Image analysis and Spot picking

• Protein spot identification

Isoelectric Focusing

;an electrophoretic method that separates proteins

according to pI

Carrier ampholytes Instable pH gradient with time

(O.Vesterberg 1969) Thousands of amphoteric compounds

Variability among batches Hard to handle (fragile)

Immobilized pH gradients

(Bjellqvist, B et al 1982) Stable pH gradient with time

Selected monomers Engineered pH gradient Easy to handle( strong plastic backing)

CH2 CH–C–NH–R

O

Improved reproducibility

2D electrophoresis

• Cell disruption

• Protein solubilization

• Prefractionation (optional)

• Isoelctric focusing (IEF)

• Equilibrium of IEF strip

• SDS PAGE

• Detection of protein spots

• Image analysis and Spot picking

• Protein spot identification

Equilibrium of IEF strip

; to allow the separated proteins to fully interact with SDS

; urea and glycerol – improves transfer of protein from IEF strip to SDS gel

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE)

SDS

The SDS molecules interact with the proteins to give rod-like complexes, containing a constant ratio of SDS per

migration.

Detection of protein spots

Non-fluorescence Dye

Coomassie Brilliant Blue

• Detection limits ~ 1μg protein

• Variability from destaining, high background

• Dynamic linear range: 1 order (10-30)

Colloidal Coomassie Brilliant Blue-G

• Increased sensitivity ( ~20 ng protein)

Silver Stain

• Sensitivity: 1-5 ng protein

• Linear Range: 10-fold

• Not compatible with downstream analysis

Selective staining dyes

Pro-Q Diamond –PhosphoproteinsPro-Q Emerald –GlycoproteinsPro-Q Amber –Transmembrane ProteinsPro-Q Sapphire –Polyhistidine Proteins

2-D Differential In-Gel Electrophoresis (2D DIGE)-pre-labeling of samples

No internal control

With internal control

Internal Control

GE Healthcare

Minimal dye labeling

• labeled 1-2 % of lysine residue of protein

• Dye charge matched to preserve pI

• molecular weight of cy dyes matched (cy2=cy3=cy5), approximately 500 Da

Saturation Dye for scarce samples

GE Healthcare

•High sensitivity ;Minimal Dyes, 0.25-1 ng protein

;Saturation Dyes, 0.01 ng for BSA

•Broad linear range ;3-5 orders of magnitude

•Easy to overlay/compare gels

•Minimizes spot pattern variability and the number of gels in

an experiment while providing simple, accurate and

reproducible spot matching.

2D Electrophoresis

• Parallel comparison

• provide map of intact proteins which reflects changes in

protein expression, isoforms or post translational modification

• resolve more than 5000 protein spots simultaneously (~2000 routinely)

• able to resolve proteins with pI around 0.001 pH units

• detect and quantify <1ng of protein per spot

• good for identifying novel proteins

Limitations

• cannot handle extremely acidic / basic proteins

• misses some large proteins & membrane proteins