Download Information Encoding in Biological Molecules: DNA and protein

Electrophoresis and 2D Gel Analysis
What is Electrophoresis ?
The migration of a charged particle (protein) through a
separation matrix toward the opposite charged
electrode
Anion
Cation
Cathode
Anode
Types of Electrophoresis
1D Gel Electrophoresis- DNA, RNA, and Protein
Agarose –low resolution, general use for RNA and DNA
Polyacrylamide – High resolution
Size determination, DNA Sequencing, gel shift, etc
SDS-PAGE-Protein
Pulse Field- DNA separation of very large fragments of genomic DNA
2D Gel Electrophoresis
Isoelectric focus and PAGE –Protein
DIGE-Differential gel electrophoresis
Two samples labeled with different fluorophores are mixed and run
on a single 2d gel
Capillary Electrophoresis (CE)
Linearized polyacrylamide, PVP
Automated DNA Sequencing, genotyping
Micro-channel/ Micro-fluidic Electrophoresis
Lab-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
slower migration
faster migration
(-)
Electrophoresis and Biomolecules
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
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
Proteins:
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 gradient
That 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
IEF apparatus from Bio-Rad
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.
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
_
SO4 Na +
Sodium Dodecyl Sulfate
C
A
T
H
O
D
E
_
_
_
_
_
_
_
_ _ _ _ _ _
_ _
_
_ _
_
_
_
_ _ _
_ _ _
_ _ _ _
_
_
_ _
_
+
+
+
+
+
+
+
A
N
O
D
E
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
Glycerol
Provides density for gel loading
Bromophenyl blue
A tracking dye used for visualization purposes during a run
Components of SDS PAGE Gel
Stacking Gel
Is 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 Gel
Small pore polyacrylamide gel (3 30% acrylamide monomer) typically
made using a pH 8.8 Tris/HCl
buffer.
Resolves protein ~24 – 205 kDa
Running Buffer
Tris/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.
Polyacrylamide Chemistry
Acrylamide monomer
N,N'-methylene-bis-acrylamide
Tetramethylethylene
diamine [TEMED]
Catalyst {O2 Scavenger}
Ammonium persulfate
{Free radical generator}
Polyacrylamide
Pore size of gel is
determined by total amount
of acrylamide and bisacrylamide
TEM image
Affect of % Acrylamide
200
200
200
45
200
45
45
200
45
6.5
45
Staining Polyacrylamide Gels
Coomassie Blue Stain- can usually detect a 10-50 ng protein per band
Blue Safe Stains -Similar to Coomassie. Destaining optional or water rinse. (8 ng/band)
BioSafe Blue
SimplyBlue
GelCode
InstantBlue-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 solution
Rinse with water
Develop with formaldehyde and carbonate followed by stopping with Glacial acetic
Fluoresecnt Stains –almost as sensitive as Silver but requires excitation
source
Flamingo Fluorescent Gel Stain
Deep Purple* Total Protein Stain
SYPRO* Ruby Protein Gel Stain
Krypton Protein Stain
IR 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 [-]
Electrostatic
Ionic
Van der Waals
Hydrophobic
Destaining Gels
Most 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 destaining
Usually requires acetic acid , methanol, and water
Safe Blue destaining
Usually requires water rinse
Silver Stains
Some methods use Potassium Ferricynide -Sodium Thiosulfate solutions
Some 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 hundreds
of 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 electrophoresis
Protein 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 Solubilization
2-20 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
3-10
4-8
5-7
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 groups
Protein will migrate in the Matrix and will find their pH equilibrium (pI)
The Second Dimension …Running the Gel
SDS Gel
pH
Similar pI
but
different mw
3
Negative electrode
4
5
6
7
8
Positive electrode
9
10
IPG strippressed
down into
the SDSPAGE gel
Similar mw but
different pI
Different IPG pH ranges yield Different
Results
pH 4
pH 4
pH 5
pH 5
pH 7
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 reagent
negative stain
Zinc stain
5-15
Reversible, simple, fast
high 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
differenceNeed to
extract spot
for MS
Extracting a Gel Spot
Cut out spot
Trypsin Digestion of
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 liver
Cy5--Tumor
Both
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