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Lehrstuhl für Siedlungswasserwirtschaft

Ingenieurfakultät Bau Geo Umwelt


Technische Universität München

Electrophoresis

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis

Electrophoresis is a technique to separate charged analytes like DNA or proteins in an


electric field. Usually a gel is prepared in which the analytes of interest travel at a
specific speed according to their charge, their size and the size of the gel pores.
Furthermore the charge of an analyte may be positive or negative, which is related to
the buffer in use and the pH within the system. Electrodes are attached to both ends of
the distance to be travelled by the analytes and an electric current is applied. If the
system is arranged so that negatively charged analytes (anions) travel towards the
anode, the system is called „anionic“ and vice versa.

Usually acrylamide gels are used for protein analysis whereas agarose or agarose-
acrylamide mixture gels have larger pore sizes and are used for the separation of
nucleic acids such as DNA or RNA.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
One can distinguish between „tube“/„strip“ and „slab“ gel electrophoresis. The former is
a tubular gel formed in glass tubes or is provided in the form of a strip, while in the
latter case a flat gel is used.The employment of tube gels has the advantage of
reduced lateral movement of the analytes. With slab gels however multiple samples
can be analyzed in parallel, which is important in order to avoid variations between
experimental conditions and therewith the experimental outcome.

Tube gel Slab gel


or strip gel or

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Western Blot (1)

Western Blot is used to detect specific proteins in a sample of e.g. prepared tissue.
The first step involves the gel electrophoretic separation of native or denatured
proteins by means of an electric field, e.g by SDS page or isoelectric focussing. Most
commonly a polyarylamide gel is employed. The separation of proteins by charge
and/or molecular weight is followed by the proteins transfer to a membrane
(nitrocellulose of PVDF), thus retaining their gel electrophoretic separation pattern.

Specific antibodies are then employed to detect and visualize the protein of interest
(target protein). This can either be done by colorimetric, chemiluminescent, radioactive
or fluorescent detection.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Western Blot (2)
Gel electrophoresis Blotting (from gel to membrane)
Ladder with reference masses
Samples Cathode
(-)

Filter
-
200kDa
Gel
180kDa
Membrane
120kDa
Filter
50kDa
30kDa
25kDa Anode
15kDa (+)
+
Polyacrylamide slab gel
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Western Blot (3)
Prior to adding the antibody, which is specific to the target protein of interest, the membrane is
incubated in a protein solution (usually bovine serum albumine, BSA) or milk to avoid non-specific
antibody binding. The first step of specific protein detection involves the addition of a „primary
antidody“, which binds to the target protein. Secondly, the unbound primary antibody is removed,
followed by the addition of a secondary antibody, which binds to the primary antibody. The
secondary antibody is linked to a reporter enzyme (e.g. horseradish preoxidase). The reporter
enzymes then cleaves e.g. a chemiluminescent agent, whereupon luminescense can be detected.

substrate product
 detectabel signal
Enzyme

Secondary antibody

Primary antibody

PD Dr. J. Graßmann; PD Dr. T. Letzel


Target protein
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis

Nothern/Southern/Eastern Blot

As opposed to Western blot, which is used for the analysis of proteins, Northern and Southern blot
are employed for the detection of RNA and DNA respectively. RNA and DNA are usually separated
with agarose gels.

The target RNA is made visible by the addition of complementary RNA („Probes“), which
specifically binds the RNA of interest. Those probes are either labeled with radioactive isotopes or
(comparable to Western blots) with a reporter enzyme like horseradish peroxidase, which
generates a detectable signal through degradation of its substrate.

The detection of DNA with Southern blot initially entails the cutting of DNA into small fragments,
followed by the electrophoretic separation. After denaturation of the DNA into single strands it is
transferred to a membrane. Comparable to Northern blot the membrane is incubated with labelled
probes specifically binding a target DNA sequence.

Eastern blot is employed for the analysis of post-translational modification of proteins, i.e.
carbohydrates. For this purpose probes are used, which specifically detect those modifications.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Native Page
Native Page is the gel electrophoretic separation of native non-degraded proteins (in contrast to SDS Page,
where proteins are denatured in the presence of SDS). Due to the maintenance of the proteins three-
dimensional structure, the separation is not only based on the protein mass but also on its charge and spacial
extent. The protein charge depends on the amino acid composition and the protein´s post-translational
modifications and is furthermore affected by the pH of the employed buffer (compare slides „Polarity and
Solubility“). Oligomeric proteins are larger than monomers and would therefore travel slower within the gel. Due
to the maintenance of the protein structure, native page allows the study of charge state alteration, which may
be associated with degradation. Different protein conformations and the association of different protein subunits,
up to the binding of protein ligands, can be studied.
Factors affecting the separation using „Native Page“ Proteins can even be recovered after separation in
Spacial extent of protein/ their native state to be used for further experiments.
Number of subunits One variant of native page is the so-called blue
+
- Charge of post-translational
-
native Page, where a blue dye (Coomassie Brilliant
modification, e.g. Sialic acid Blue) is added to the sample to provide a charge to
„overwhelm“ the proteins intrinsic charge. The
- - separation is then based on the assumption of the
+ + bound dye to be proportional to the protein´s mass
- - -
pH of buffer
(comparable to SDS Page). However the added dye
++ - might cause protein denaturation.
Surface charge

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
SDS- Page

SDS- page, which is performed using polyacrylamide gels, is a common method for the separation of proteins.
Due to the denatured state of the proteins (in contrast to native page), this method allows the estimation of the
molecular weight of proteins or individual protein subunits. The denaturation of proteins is caused by the
presence of the anionic detergent SDS (sodium dodecyl sulphate) and additional reducing agents to break
disulfide bridges, which stabilize the native protein conformation. SDS then coats the unfolded protein, therewith
providing negative charges. The quantity of attached SDS molecules and therefore the
number of charges is proportional to the length of the unfolded protein´s
peptide chain. Thus, the proteins are separated according to their
size in an electric field.

„Ladder“ of known Unknown samples


molecular masses

200kDa
180kDa

120kDa Estimated molecular weight: 40kDa

50kDa
30kDa
25kDa Estimated molecular weight: 27kDa

PD Dr. J. Graßmann; PD Dr. T. Letzel 15kDa


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Detergent (1)
Detergents are amphiphilic molecules, i.e. consisting of a hydrophilic and a hydrophobic part. They are able to
align so as to form micelles with the hydrophobic part of the detergent orientated to the inside of the micelle to
avoid interaction with water. Critical micellar concentration (cmc) is the concentration above which detergents
form micelles in water.

Detergents are used for the solubilization of hydrophobic molecules, for the disruption of cell membranes, the
denaturation of proteins (e.g. SDS) or the isolation of transmembrane proteins, which contain hydrophobic areas
physiologically located within the cell membrane.
General structure of detergents, e.g. SDS
Hydrophobic chain Hydrophilic head

Concentration
above cmc

Drawing of chemical structures was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Detergent (2)
Proteins are folded so as to shield hydrophobic parts of the peptide chain from the surrounding aqueous milieu.
The protein surface consequently mainly consists of hydrophilic amino acid side chains. The proteins native
conformational state is furthermore stabilized by hydrogen bondings and disulfide bridges. Due to its amphiphilic
structure, SDS is able to coat the peptide chain to shield the hydrophobic amino acid side chains (hydrophobic
interaction between hydrophobic amino acids and hydrophobic SDS chain), whereas the hydrophilic head of SDS
is oriented towards the aqueous environment. This ultimatively causes the protein to unfold.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Isoelectric focussing (1)

Protein separation in an electric field, which is based on the specific isoelectric point of proteins is
called isoelectric focussing. Proteins consist of a succession of amino acids, each of them
potentially carrying positive and negative charge(s). Those charges are affected by the pH value
of the surrounding milieu. At a specific pH the overall charge of the amino acids is affected so as
to the protein eventually carries no net charge. At a pH below the pI, the protein is positively
charged. At a pH above the pI, it will carry a surplus of negative charges.

e.g. amino acid alanine (pI = 6.0)

+ +
NH3 NH3 NH2
- -
H3C C COOH H3C C COO H3C C COO
H H H
cation zwitterion anion
pH < pI pH = pI pH > pI

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Isoelectric focussing (2)
A peptide, which is composed of several amino acids might not have a net charge at its isoelectric
point, but nevertheless contains negatively or positively charged groups, which neutralize each
other.
H H H H H H H H H
+ NH C C N C C N C C N C C N C COO
-
3

CH3 O CH2 O CH2 O CH2 O CH2


NH
COO
- CH2 CH2
COO
- + NH
CH2

CH2
+ NH3

3 negative charges + 3 positive charges = zero net charge


PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Isoelectric focussing (3)

Empty strip holder

Protein sample is
loaded

pH 2 3 4 5 6 7 8 9 10 11 12
IF strip with pH
gradient is placed
into the strip holder
PD Dr. J. Graßmann; PD Dr. T. Letzel
Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Isoelectric focussing (4)

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
2D-Electrophoresis

2D electrophoresis of proteins consists of two independent electrophoresis steps:

The first one separates the proteins according to their isoelectric point using a strip gel
with an immobilized pH gradient. The method is called isoeletric focussing (IF). The
isoelectric point is the pH value where the protein carries no net charge. At that specific
pH the protein therefore remains unaffected by the electric current applied, i.e. it stops
traveling through the gel.

After IF, a second (usually polyacrylamide) gel is used to further separate the proteins,
e.g. according to their size. For the second separation step the proteins can be
denatured in the presence of SDS (sodium dodecyl sulfate). SDS is a negatively
charged detergent, which binds to the denatured proteins. Due to the charge provided
by SDS, the proteins can then be electrophoretically separated according to their size.
They are visualized by staining, either with Coomassie Blue or silver.

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
2D-Electrophoresis

PD Dr. J. Graßmann; PD Dr. T. Letzel


Lehrstuhl für Siedlungswasserwirtschaft
Ingenieurfakultät Bau Geo Umwelt
Technische Universität München

Electrophoresis
Polyacrylamide gel
Acrylamide monomers polymerize in the presence of bisacrylamid, forming a three-dimensional
network through cross-linking. The size of the pores within the gel can be regulated by the ratio of
acrylamide to bisacrylamide. The gel composition can be adjusted depending on the protein size to
be analyzed. Lower percentage of acrylamide is generally used for the separation of high
molecular weight proteins and vice versa. Due to the formation of tunnels with different diameters,
small proteins travel faster within the electric field applied.

Drawing of chemical structures was performed with „MarvinSketch 14.8.25.0“, ChemAxon (www.chemaxon.com)
PD Dr. J. Graßmann; PD Dr. T. Letzel