Download Prezentace aplikace PowerPoint

ELECTROPHORETIC METHODS
Basic principles of electrophoresis
1. It is the process of moving charged
biomolecules in solution by applying
an electrical field across the mixture.
2. Biomolecules moved with a speed
dependent on their charge, shape,
and size and separation occures on
the basis of molecular size.
Electrophoresis is used: for analysis and
purification of very large molecules
(proteins, nucleic acids)
for analysis of simpler charged
molecules (sugars, amino acids,
peptides, nucleotides, and simpler
ions).
When charged molecules are
placed in an electric field,
they migrate toward either
the positive (anode) or
negative (cathode) pole
according to their charge.
1.
2.
3.
4.
Factors influenced
electrophoresis mobility:
net charge of the
molecule
size and shape
concentration of the
molecule in solution
Amino acids have characteristic titration curves
Proton
donor
Proton
acceptor
At the midpoint – pK=9.60 there
is equimolar concentration of
proton donor and proton
acceptor.
+
Izoelectric point
Dipolar ion
At the midpoint – pK1=2.34 there
is equimolar concentration of
proton donor and proton acceptor.
+
Fully protonated
form at wery low pH
Proton
donor
Proton
acceptor
Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry
Electrophoresis is carried out by applying a thin
layer
Aqueous protein solution is immobilized in a solid hydrophilic
support.
Solid matrix with pores which are used:
• paper
• starch
• cellulose acetate
• polyacrylamide
• agar/agarose
Molecules in the sample move through porous matrix at
different velocity.
• Electrophoresis can be one
dimensional (i.e. one plane of
separation) or two dimensional.
• One dimensional electrophoresis is
used for most routine protein and
nucleic acid separations. Two
dimensional separation of proteins is
used for finger printing , and when
properly constructed can be
extremely accurate in resolving all of
the proteins present within a cell
(greater than 1,500).
• Most common stabilizing media are
polyacrylamide or agarose gels.
Buffers
• Function of buffer
1. carries the applied current
2. established the pH
3. determine the electric charge on the solute
• High ionic strength of buffer
– produce sharper band
– produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
Zone electrophoresis
•
•
•
•
Much simple method
Much greater resolution
Require small sample
Acetate cellulose – support medium
Protein separation depends on :
• Type and number of ionizable side chains of amino
acids - R.
• Size of net charge (positive or negative).
• Negatively charged proteins move towards the anode.
• Positively charged proteins move towards the
cathode.
Stripe of cellulose acetate
Electrophoresis
Major protein components
separate into discrete zones
Densitometer tracing
density of zones is proportional
to the amount of protein
Example of application of zone electrophoresis in
clinical practice
Hypergamaglobulinemia
Hypogamaglobulinemia
Normal serum
Gel electrophoresis
• Gel is a colloid in a solid form (99% is water).
• Gel material acts as a "molecular sieve.
• During electrophoresis, macromolecules are forced to
move through the pores when the electrical current is
applied.
Support media
• Agarose and polyacrylamide gels are acrosslinked, spongelike structure
• It is important that the support media is
electrically neutral. Presence of charge group
may cause:
-Migration retardation
-The flow of water toward one or the other electrode so
called ‘Electroendosmosis (EEO)’, which decrease
resolution of the separation
Agarose – highly purified
polysaccharide derived from
agar (extracted from
seeweed), long sugar
polymers held together by
hydrogen and hydrophobic
bonds.
Acrylamide (CH2=CH-CONH2)
Polyacrylamide gel structure
held together by covalent
cross-links
Agarose gels
• For the separation of (1) large protein or protein
complex (2) polynucleotide 50-30,000 base-pairs
• The pore size is determined by adjusting the
concentration of agarose in a gel (normally in the rank
of 0.4-4%
OH
O
CH2OH O
OH
O
O
OH
O
O
Polyacrylamide gels
CH2=CHCONH2
Acrylamide
+
CH2(NHCOHC=CH2)2
N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CHCO
CO
CO
NH
NH2
NH
n
CH2
CH2
NH
NH2 NH
CO
CO
CO
-CH2-CH-CH2-CH-CH2-CHn
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
•
•
•
•
SDS (also called lauryl sulfate) - anionic detergent
Molecules in solution with SDS have a net negative charge within a wide pH
range.
A polypeptide chain binds amounts of SDS in proportion to its relative
molecular mass.
The negative charges on SDS destroy most of the complex structure of
proteins, and are strongly attracted toward an anode (positively-charged
electrode) in an electric field.
Diagrams of vertical slab gel assembly
Determination of molecular mass
Commonly used protein stains
Stain
Detection limit
Ponceau S
1-2 mg
Amido Black
1-2 mg
Coomassie Blue
1.5 mg
India Ink
100 ng
Silver stain
Colloidal gold
10 ng
3 ng
Staining with Coomasie blue
1
2
3
1
Assesment of individual lines
2
3
An ethidium-stained gel photographed under UV light
**Each band that you see is a collection of millions of
DNA molecules, all of the same length!!
Western blott technique
• Western blot (also called immunoblot)
is a technique to detect specifically one
protein in a mixture of large number of
proteins and to obtain information about
the size and relative amounts of the
protein present in different samples.
• In first proteins are separated using
SDS-polyacrylamide gel electrophoresis.
• Then they are moved onto a
nitrocellulose membrane. The proteins
retain the same pattern of separation
they had on the gel.
• An antibody is then added to the
solution which is able to bind to its specific
protein and forms an antibody-protein
complex with the protein of interest. (In
fact there is no room on the membrane for
the antibody to attach other than on the
binding sites of the specific target protein).
• Finally the nitrocellulose membrane is
incubated with a secondary antibody,
which is an antibody-enzyme conjugate
that is directed against the primary
antibody.
• The location of the antibody is revealed
by incubating it with a substrate that the
attached enzyme converts to a product
that can be seen and followed and then
photographed.
Isoelectric focusation
Proteins are separated in pH gradient.
Protein migrate into the point where its net charge is zero –
isoelectric pH.
Protein is positively charged in solutions at pH values below its pI.
Protein is negatively charged in solution at pH above its pI.
Two-dimensional gel electrophoresis
(2-D electrophoresis )
 In the first dimension, proteins are resolved in according to their isoelectric
points (pIs) using immobilized pH gradient electrophoresis (IPGE), isoelectric
focusing (IEF), or non-equilibrium pH gradient electrophoresis.
 In the second dimension, proteins are separated according to their
approximate molecular weight using sodium dodecyl sulfate poly-acrylamideelectrophoresis (SDS-PAGE).
Electrophoreogram of the mixture of proteins
Protein „maps“ are compare with control pattern of normal
healthy person and abnormalities are analysed
Capillary electrophoresis
Capillaries are typically of 50 µm inner diameter and 0.5 to 1 m in
length.
Due to electroosmotic flow, all sample components migrate towards
the negative electrode.
The capillary can also be filled with a gel, which eliminates the
electroosmotic flow. Separation is accomplished as in conventional gel
electrophoresis but the capillary allows higher resolution, greater
sensitivity, and on-line detection.
Electroosmotic flow
The surface of the silicate glass capillary contains negatively-charged
functional groups that attract positively-charged counterions. The
positively-charged ions migrate towards the negative electrode and
carry solvent molecules in the same direction. This overall solvent
movement is called electroosmotic flow. During a separation, uncharged
molecules move at the same velocity as the electroosmotic flow (with
very little separation). Positively-charged ions move faster and
negatively-charged ions move slower.