2. Electrophoresis
2
Electrophoresis is a method whereby charged molecules in solution, mainly proteins and nucleic
acids, migrate in response to an electrical field.
Their rate of migration through the electrical field, depends on:
- the strength of the field,
- on the net charge, size,
- and shape of the molecules,
- and also on the ionic strength, viscosity, and temperature of the medium in which the
molecules are moving.
As an analytical tool, electrophoresis is simple, rapid and highly sensitive.
It can be used analytically to study the properties of a single charged species or mixtures of
molecules. It can also be used preoperatively as a separating technique
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3. Electrophoresis
3
Electrophoresis is usually done with gels formed in tubes, slabs, or on a
flat bed.
In many electrophoresis units, the gel is mounted between two buffer
chambers containing separate electrodes, so that the only electrical
connection between the two chambers is through the gel.
Separation according to migration of charged particles in electric field
Electrolysis: Chemical decomposition supplemented by current (components
reach electrode)
F = v f = q E
V=Velocity, E=Electric field, q=Charge f=Frictional coefficient
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4. Electrophoretic mobility
4
Electrophoretic mobility μ = v / E = q / f
V=Velocity, E=Electric field, q=Charge f=Frictional
coefficient
Depends on
Particle property :Surface, charge, density & size
Solution properties: Ionic strength, pH,
Conductivity,viscosity
Temperature & Voltage
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5. In most electrophoresis units, the gel is mounted between two buffer
chambers containing separate electrodes so that the only electrical
connection between the two chambers is through the gel.
5 Asheesh Pandey
10. Interrelation of Resistance,
Voltage, Current and Power
10
Two basic electrical equations are important in
electrophoresis
The first is Ohm's Law, I = E/R
The second is P = EI
This can also be expressed as P = I2
R
In electrophoresis, one electrical parameter, either current,
voltage, or power, is always held constant
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11. Consequences
11
Under constant current conditions (velocity is directly
proportional to current), the velocity of the molecules is
maintained, but heat is generated.
Under constant voltage conditions, the velocity slows, but no
additional heat is generated during the course of the run
Under constant power conditions, the velocity slows but
heating is kept constant
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12. The Net Charge is Determined by
the pH of the Medium
12
Proteins are amphoteric compounds, that is, they contain
both acidic and basic residues
Each protein has its own characteristic charge properties
depending on the number and kinds of amino acids carrying
amino or carboxyl groups
Nucleic acids, unlike proteins, are not amphoteric. They
remain negative at any pH used for electrophoresis
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13. Temperature and Electrophoresis
13
Important at every stage of
electrophoresis
During Polymerization
Exothermic Reaction
Gel irregularities
Pore size
During Electrophoresis
Denaturation of proteins
Smile effect
Temperature Regulation of Buffers
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14. What is the Role of the Solid
Support Matrix?
14
It inhibits convection and diffusion, which
would otherwise impede separation of
molecules
It allows a permanent record of results
through staining after run
It can provide additional separation through
molecular sieving
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15. Agarose and Polyacrylamide
15
Although agarose and polyacrylamide differ greatly in their
physical and chemical structures, they both make porous
gels.
A porous gel acts as a sieve by retarding or, in some cases, by
completely obstructing the movement of macromolecules
while allowing smaller molecules to migrate freely.
By preparing a gel with a restrictive pore size, the operator
can take advantage of molecular size differences among
proteins
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16. Agarose and Polyacrylamide
16
Because the pores of an agarose gel are
large, agarose is used to separate
macromolecules such as nucleic acids, large
proteins and protein complexes
Polyacrylamide, which makes a small pore
gel, is used to separate most proteins and
small oligonucleotides.
Both are relatively electrically neutral
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17. Agarose Gels
17
Agarose is a highly purified uncharged polysaccharide derived from
agar
Agarose dissolves when added to boiling liquid. It remains in a liquid
state until the temperature is lowered to about 40° C at which point
it gels
The pore size may be predetermined by adjusting the concentration
of agarose in the gel
Agarose gels are fragile, however. They are actually hydrocolloids,
and they are held together by the formation of weak hydrogen and
hydrophobic bonds
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18. Structure of the Repeating Unit of
Agarose, 3,6-anhydro-L-galactose
18
Basic
disaccharide
repeating units of
agarose,
G: 1,3-β-d-
galactose
and
A: 1,4-α-l-3,6-
anhydrogalactoseAsheesh Pandey
20. Polyacrylamide Gels
20
Polyacrylamide gels are tougher than agarose gels
Acrylamide monomers polymerize into long chains
that are covalently linked by a crosslinker
Polyacrylamide is chemically complex, as is the
production and use of the gel
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22. Components of Lysis Buffer
22
Buffer (Tris or Hepes buffer with pH 7-8)
Salt (usually NaCl 150mM (low) to 500mM(high)
Chelating agent (EDTA or EGTA)
Detergent
Protease inhibitor
Phosphatase inhibitor (optional)
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23. Lysing Cells
23
Treat cells with appropriate conditions depending on
the experiment
Pellet cells and lyse them in the appropriate lysis
buffer.
Most important ingredient in lysis buffer is detergent.
Most stringent to weakest
SDS
NP-40
Triton X-100
Tween 20
Digitonin
CHAPS
Brig
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24. 24
Second most important ingredient is protease
inhibitors.
Once proteins are denatured by detergents, they are
susceptible to degradation by proteases.
Need more than one inhibitor since there are lots of
proteases.
Protease inhibitors
Aprotinin
Leupeptin
PMSF (phenylmethylsulfonyl fluoride)
Add immediately before lysis since PMSF activity decreases over
time in aqueous solutions. About 15-30 minutes of activity.
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25. Phosphatase inhibitors
25
Need to add to lysis buffer if using
phopshospecific antibodies or suspect protein of
interest is phosphorylated
Inhibitors
ZnCl2
NaF
Na-Vanadate (tyrosine phosphatase inhibitor, add prior
to lysis since only active in pH 7 for minutes)
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26. Further denature proteins
26
Add lysate either a known concentration of proteins
or cell number equivalent to SDS loading buffer
SDS Loading Buffer
Buffer (Tris-Cl pH 6.0)
2% SDS
0.1% bromophenol blue
10% glycerol (allows sol’n to sink to bottom of gel wells)
β-mercaptoethanol ( reducing agent)
SDS loading gel mixed with lysate is boiled to further
denature proteins. (104gm SDS/1gm Protein)
1:10 ratio loading buffer to lysate
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27. Ingredients in Gel
27
Sodium dodecyl sulfate (SDS)
Tris buffer (either glycine or tricine)
Acrylamide and NN-bis-acrylamide
Forms gel matrix
TEMED
Catalyst for polymerization (produces free radials from APS)
Ammonium persulfate (APS)
Source of free radials for polymerization
Could purchase pre-cast gels if you have the money.
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28. Pouring your gel
28
Pour running gel first. Contains Tris buffer at pH 8.0.
The percent of acrylamide may be adjusted for better
resolution of proteins (5-15%)
Pour ethanol or distilled water on top of gel for even
polymerization.
Leave enough room to pour stacking gel, about one forth
of total gel.
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29. Pouring gel continues
29
After the running gel has polymerized, the gel is
washed with distilled water to remove any debris
on the gel to give a good interface between
stacking and running gels. The stacking gel is
pour. It contains Tris buffer at pH 6.8.
5% acrylamide for maximum porosity
Deposits proteins on stacking and running gel interface
which concentrates the proteins and provides better
resolution.
Insert comb to form wells at an angle to prevent air
bubbles.
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30. Loading gel
30
Remove comb and wash wells out with running
buffer.
Best to use loading tips (Hamilton syringes also
work) to load samples.
Start at the bottom of well and work your way up
the well.
Glycerol in the loading buffer will keep sample in
well.
Optional- Running buffer left in wells or wells
empty.
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31. Running Gel
31
After loading samples, added running buffer to
upper reservoir and lower reservoir. Hint. Add
upper reservoir first to detect leaks.
Running buffer provides the ions to conduction the
current through the gel.
SDS makes proteins negatively charged that
attaches the proteins to the anode.
Therefore in electrophoresis, the current must run
from cathode (negatively charged, black) to the
anode (positive charged, red).
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32. Considerations with PAGE
32
Preparing and Pouring Gels
Determine pore size
Adjust total percentage of acrylamide
Vary amount of crosslinker
Remove oxygen from mixture
Initiate polymerization
Chemical method
Photochemical method
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33. Considerations with PAGE
33
Analysis of Gel
Staining or autoradiography followed by
densitometry
Blotting to a membrane, either by capillarity or
by electrophoresis, for nucleic acid
hybridization, autoradiography or
immunodetection
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34. SDS Gel Electrophoresis
34
In SDS separations, migration is determined not by intrinsic
electrical charge of polypeptides but by molecular weight
Sodium dodecylsulfate (SDS) is an anionic detergent which
denatures secondary and non–disulfide–linked tertiary
structures by wrapping around the polypeptide backbone. In so
doing, SDS confers a net negative charge to the polypeptide in
proportion to its length
When treated with SDS and a reducing agent, the polypeptides
become rods of negative charges with equal “charge densities"
or charge per unit length.
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36. Continuous and Discontinuous Buffer
Systems
36
A continuous system has only a single separating
gel and uses the same buffer in the tanks and the
gel
In a discontinuous system a nonrestrictive large
pore gel, called a stacking gel, is layered on top of
a separating gel
The resolution obtainable in a discontinuous system
is much greater than that obtainable in a
continuous one. However, the continuous system is
a little easier to set up
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38. Commonly used protein stains
38
Stain Detection limit
Ponceau S 1-2 µg
Amido Black 1-2 µg
Coomassie Blue 1.5 µg
India Ink 100 ng
Silver stain 10 ng
Colloidal gold 3 ng
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41. Determining Molecular Weights of
Proteins by SDS-PAGE
Run a gel with standard proteins of known
molecular weights along with the
polypeptide to be characterized
A linear relationship exists between the
log10 of the molecular weight of a
polypeptide and its Rf
Rf = ratio of the distance migrated by the
molecule to that migrated by a marker dye-
front
The Rf of the polypeptide to be
characterized is determined in the same
way, and the log10 of its molecular weight is
read directly from the standard curve
41Asheesh Pandey
42. Blotting
42
Blotting is used to transfer proteins or
nucleic acids from a slab gel to a membrane
such as nitrocellulose, nylon, DEAE, or CM
paper
The transfer of the sample can be done by
capillary or Southern blotting for nucleic
acids (Southern, 1975) or by
electrophoresis for proteins or nucleic
acids
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43. Isoelectric Point
There is a pH at which there is no net
charge on a protein; this is the
isoelectric point (pI).
Above its isoelectric point, a protein has
a net negative charge and migrates
toward the anode in an electrical field.
Below its isoelectric point, the protein is
positive and migrates toward the
cathode.
43Asheesh Pandey
44. Isoelectric Focusing
44
Isoelectric focusing is a method in which proteins
are separated in a pH gradient according to their
isoelectric points
Focusing occurs in two stages; first, the pH
gradient is formed
In the second stage, the proteins begin their
migrations toward the anode if their net charge is
negative, or toward the cathode if their net
charge is positive
When a protein reaches its isoelectric point (pI) in
the pH gradient, it carries a net charge of zero
and will stop migrating
Difference 2-D?
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45. Two-Dimensional Gel Electrophoresis
45
Two-dimensional gel electrophoresis is widely
used to separate complex mixtures of proteins
into many more components than is possible in
conventional one-dimensional electrophoresis
Each dimension separates proteins according
to different properties
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46. 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-acrylamide-electrophoresis
(SDS-PAGE).
46 Asheesh Pandey
47. O’Farrell 2D Gel System
47
The first dimension tube gel is
electrofocused
The second dimension is an SDS slab gel
The analysis of 2-D gels is more
complex than that of one-dimensional
gels because the components that show
up as spots rather than as bands must
be assigned x, y coordinates
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48. • DIGE can be done in one-or two-dimensions. Same
principle.
• Requires fluorescent protein stains (up to three of
these), a gel box, and a gel scanner.
• Dyes include Cy2, Cy3 and Cy5 (Amersham system).
• These have similar sizes and charges, which means
that individual proteins move to the same places on
2-D gels no matter what dye they are labeled with.
• Detection down to 125 pg of a single protein.
DIfference Gel ElectrophoresisDIfference Gel Electrophoresis
DIGEDIGE
48 Asheesh Pandey
50. • After running the gels, three scans are done to extract
the Cy2, Cy3, and Cy5 fluorescence values.
• Assuming the Cy2 is the internal control, this is used to
identify and positionally match all spots on the different
gels.
• The intensities are then compared for the Cy3 and Cy5
values of the different spots, and statistics done to see
which ones have significantly changed in intensity as a
consequence of the experimental treatment.
50 Asheesh Pandey
51. • Labeling slightly shifts the masses of the proteins, so to cut
them out for further analysis, you first stain the gel with a
total protein stain.
• SYPRO Ruby is used for this purpose (Molecular Probes).
• When designing 2-D DIGE experiments, the following
recommendations should be considered:
1. Inclusion of an internal standard sample on each gel.
These can comprise a mixture of known proteins of
different sizes, or simply a mixture of unknown proteins
(one of your samples).
2. Use of biological replicates.
3. Randomization of samples to produce unbiased results.
51 Asheesh Pandey
53. Blotting: History
53
Southern Blotting is named after its
inventor, the British biologist Edwin
Southern (1975)
Other blotting methods (i.e. western blot,
WB, northern blot, NB) that employ
similar principles, but using protein or
RNA, have later been named in reference
to Edwin Southern's name.
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54. SOUTHERN BLOTTING ?
54
Experimental procedure
DNA is extracted from cells, leukocytes.
DNA is cleaved into many fragments by
restriction enzyme (BamH1, EcoR1 etc)
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56. 56
The resulting fragments are separated on the basis of
size by electrophoresis.
The large fragments move more slowly than the
smaller fragments.
The lengths of the fragments are compared with band
of relative standard fragments of known size.
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57. 57
The DNA fragments are denatured and transferred to
nitrocellulose membrane (NYTRAN) for analysis.
DNA represents the individual's entire genome, the
enzymic digest contains a million or more fragments.
The gene of interest is on only one of these pieces of
DNA.
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58. 58
DNA segments were visualized by a nonspecific
technique, they would appear as an unresolved blur of
overlapping bands.
To avoid this, the last step in Southern blotting uses a
probe to identify the DNA fragments of interest.
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59. 59
Southern blot analysis depend on the specific
restriction endonuclease
The probe used to visualize the restriction fragments.
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60. •Labeled material to detect a target.
•For DNA: 20-30 nucleotides, complementary to a region in the gene
•Methods of labeling:
•Non-radioactive e.g. Biotin•Radioactive e.g. 32
P
•Sensitive
•Relatively cheap
•Hazardous
You should follow the radioactive
waste disposal regulations.
•Sensitive
•Relatively expensive
Target DNA
Probe
Biotin Avidin
*
Target DNA
Probe
*
Probes
60 Asheesh Pandey
61. The binding between ss labeled probe to a complementary
nucleotide sequence on the target DNA.
Degree of hybridization depends on method of probe labeling
(radioacitve or non-radioactive system e.g. biotin-avidin.
Hybridization
61 Asheesh Pandey
62. 62
Detection of mutations
The presence of a mutation affecting a restriction site
causes the pattern of bands to differ from those seen with
a normal gene.
A change in one nucleotide may alter the nucleotide
sequence so that the restriction endonuclease fails to
recognize and cleave at that site
(for example, in Figure, person 2 lacks a restriction site
present in person 1).
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63. Steps
Digestion of genomic DNA (w/ ≥ one RE) DNA fragments
Size-separation of the fragments (standard agarose gel electrophoresis)
In situ denaturation of the DNA fragments (by incubation @ ↑temp)
Transfer of denatured DNA fragments into a solid support (nylon or
nitrocellulose).
Hybridization of the immobilized DNA to a labeled probe (DNA, RNA)
Detection of the bands complementary to the probe (e.g. by
autoradiography)
Estimation of the size & number of the bands generated after digestion of
the genomic DNA w/ different RE placing the target DNA within a
context of restriction sites)
63 Asheesh Pandey
64. METHODS OF TRANSFER
Downward Capillary Transfer
Upward Capillary Transfer
Simultaneous Transfer to Two Membranes
Electrophoretic Transfer
Vacuum Transfer
64 Asheesh Pandey
65. Example of Transfer
Upward Capillary Transfer
Weight
Glass Plate
Whatman 3MM pape
Gel
Paper towels
Membrane (nylon
or nitrocellulose)
Whatman 3MM
paper
Transfer buffer
65 Asheesh Pandey
66. Buffer drawn from a
reservoir passes
through the gel into a
stack of paper towels
DNA eluted from
the gel by the
moving stream of
buffer is deposited
onto a membrane
weight tight connection
66 Asheesh Pandey
67. Northern Blotting
Northern Hybridization
A northern blot is a method routinely used in
molecular biology for detection of a specific RNA
sequence in RNA samples.
The method was first described in the seventies
(Alwine et al. 1977, 1979)
It is still being improved (Kroczek 1993), with the
basic steps remaining the same
67 Asheesh Pandey
68. Basis Steps of NB
1. Isolation of intact mRNA
2. Separation of RNA according to size (through a
denaturing agarose gel e.g. with Glyoxal/formamide)
Transfer of the RNA to a solid support
Fixation of the RNA to the solid matrix
Hybridization of the immobilized RNA to probes
complementary to the sequences of interest
Removal of probe molecules that are nonspecifically
bound to the solid matrix
Detection, capture, & analysis of an image of the
specifically bound probe molecules.
68 Asheesh Pandey
69. Applications
Study of gene expression in eukaryotic cells:
To measure the amount & size of RNAs
transcribed from eukaryotic genes
To estimate the abundance of RNAs
Therefore, it is crucially important to equalize
the amounts of RNA loaded into lanes of gels
69 Asheesh Pandey
70. Examples of methods to equalize the
amounts of RNA loaded into lanes of gels
OD260
Use of housekeeping gene (endogenous constitutively-
expressed gene): Normalizing samples according to their
content of mRNAs of this housekeeping gene
70 Asheesh Pandey
72. WB: Definition
Blotting is the transfer of separated proteins
from the gel matrix into a membrane, e.g.,
nitrocellulose membrane, using electro- or
vacuum-based transfer techniques.
Towbin H, et al (1979). "Electrophoretic transfer of proteinsTowbin H, et al (1979). "Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure andfrom polyacrylamide gels to nitrocellulose sheets: procedure and
some applications.".some applications.". Proc Natl Acad Sci U S A.Proc Natl Acad Sci U S A. 76 (9): 4350–435476 (9): 4350–4354
72 Asheesh Pandey
73. Applications & Advantages
Applications:
To determine the molecular weight of a protein
(identification)
To measure relative amounts (quantitation) of the
protein present in complex mixtures of proteins that are
not radiolabeled (unlike immunoprecipitation)
Advantages:
WB is highly sensitive technique
As little as 1-5 ng of an average-sized protein can be
detected by WB
73 Asheesh Pandey
74. 74
Western blotting
The main steps of blotting technique in a
chronological order will be as follows:
Blocking
Probing with the specific antibody(ies)
Wash
Detection
Washing
X-ray (Gel Documentation System)
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75. Electrophoretic Transfer: An
Overview
Important Issue:Important Issue:
Where to put the gel and the membrane relative to theWhere to put the gel and the membrane relative to the
electroblotting transfer electrodes?electroblotting transfer electrodes?
75 Asheesh Pandey
76. Direction of Transfer
Perpendicularly from the direction of travel of proteins
through the separating gel
Gel
Membrane
Probe with specific Ab
76 Asheesh Pandey
77. Factors Affecting Transfer
Efficiency
1. The Composition of the gel
2. Whether there is complete contact of the
gel with the membrane
3. The position of the electrodes
4. The transfer time
5. The size & composition of proteins
6. The field strength
7. The presence of detergents77 Asheesh Pandey
85. 85
Blocking of Blot
Several measures should be followed to
decrease the nonspecific reactions to a
minimum, i.e., increasing the signal to noise
ratio.
Blocking step is the incubation of the membrane
with solution containing BSA or fat-free milk or
casein for a sufficient time with shaking.
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87. 87
Primary Antibody labeling
The immobilized proteins on the surface of the
membrane can be detected using a specific, labeled
antibody.
Labeling of the antibody can be performed using a
radioactive or non- radioactive method.
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88. 88
Primary Antibody probing
The blot is first incubated with a primary antibody
followed by the addition of a labeled secondary antibody that
has species specificity for the primary one.
For example, probing of the membrane using mouse
primary antibody and anti- mouse secondary antibody.
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89. 89
Detection and interpretation
A prestained MW standard is included in a separate
lane during electrophoresis to allow the identification of
the MW of the target protein.
Similar to the analysis of electrophoresis results on a gel,
the data on the membrane can be quantitatively
analyzed using gel documentation system.
Asheesh Pandey
90. 90
Detection and interpretation (continue)
Quantification of a specific protein band can be
achieved by densitometry and integrating the areas under
the peaks.
Several gel documentation systems are commercially
available that can be useful for analysis of results from
the gel or membranes.
Asheesh Pandey
91. Comparison between WB & SB.
91
Similarities:
Electrophoretically separated components (proteins in
WB & DNA in SB), are transferred from a gel to a
solid support and probed with reagents that are specific
for particular sequences of AA (WB) or nucleotides
(SB).
Asheesh Pandey
92. References
92
Lippincott, Illustrated review of Biochemistry, 4th
edition
Molecular Cloning: A Laboratory Manual, J
Sambrook, EF Fritsch, T Maniatis
Catalogues of some commercial companies
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