2D Electrophoresis

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
A Guide to Protein Isolation
Author: Dennison, Clive. 2002 P 116
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.
Molecular weight (MW)
; SDS PAGE, protein sequencing

pK1=1.88
pK2=3.65
pK3=9.60
pI of aspartate = 1/2(1.88 + 3.65) = 2.77

pK1=2.32
pK2=9.60
pI of Glycine = 1/2(2.32 + 9.60) = 5.96
http://scansite.mit.edu/calc_mw_pi.html
Isoelectric point (PI)
; pH at which a molecule has no net charge.
MW calculation: MW = ΣAAi x MWi
Molecular weight (MW)
(+protein modification)
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
Cell disruption
osmotic lysis
freeze-thaw cycling
detergent lysis
enzymatic lysis of the cell wall
sonication
grinding with (or without) liquid nitrogen with mortar and pestle
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), tris(2-carboxyethyl)phosphine (TCEP)

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
R = weakly acidic or basic buffering group
Görg, A. , Nature 1991
Improved
reproducibility
Proteomics 2004, 4, 3665–3685
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
Dithiothreitol (DTT)
- reduction of (reformed)
disulphide bridges
Iodoacetamide
- alkylation of proteins
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
Görg et al., Electrophoresis 1987, 1988
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 mg of protein.
At this level the negative charge on the SDS is sufficient to mask the charge
on the protein and all proteins consequently have essentially the same
charge/mass ratio and an anodic 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
Fluorescent Dyes
Higher sensitivity
SYPRO Orange, Red (Molecular Probes) – 4-8ng
SYPRO Ruby (Molecular Probes) – ~1ng
DeepPurple (GE Healthcare) – <1ng
Krypton (Pierce Biochem) – ~0.25ng
Linear range over 3 orders of magnitude
Expensive

Selective staining dyes
Pro-Q Diamond –Phosphoproteins
Pro-Q Emerald –Glycoproteins
Pro-Q Amber –Transmembrane Proteins
Pro-Q Sapphire –Polyhistidine Proteins
2-D Differential In-Gel Electrophoresis (2D DIGE)-pre-labeling of samples
GE Healthcare
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
GE Healthcare
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