electrophoresis and blotting techniques by asheesh pandey
TRANSCRIPT
Electrophoresis and Blotting Techniques
Asheesh Pandey, IET Lucknow1
Electrophoresis
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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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Electrophoresis
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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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Electrophoretic mobility
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Electrophoretic mobility μ = v / E = q / fV=Velocity, E=Electric field, q=Charge f=Frictional
coefficientDepends on
Particle property :Surface, charge, density & sizeSolution properties: Ionic strength, pH,
Conductivity,viscosityTemperature & Voltage
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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.
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The Technique
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The Technique
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Slab Gel Unit
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Flat Bed Unit
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Interrelation of Resistance, Voltage, Current and Power
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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 = I2R
In electrophoresis, one electrical parameter, either current, voltage, or power, is always held constant
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Consequences
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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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The Net Charge is Determined by the pH of the Medium
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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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Temperature and Electrophoresis
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Important at every stage of electrophoresisDuring Polymerization
Exothermic ReactionGel irregularitiesPore size
During ElectrophoresisDenaturation of proteinsSmile effectTemperature Regulation of Buffers
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What is the Role of the Solid Support Matrix?
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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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Agarose and Polyacrylamide
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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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Agarose and Polyacrylamide
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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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Agarose Gels
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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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Structure of the Repeating Unit of Agarose, 3,6-anhydro-L-galactose
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Basic disaccharide repeating units of agarose,
G: 1,3-β-d-galactose
and
A: 1,4-α-l-3,6-anhydrogalactose
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Gel Structure of Agarose
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Polyacrylamide Gels
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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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Crosslinking Acrylamide Chains
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Components of Lysis Buffer
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Buffer (Tris or Hepes buffer with pH 7-8)Salt (usually NaCl 150mM (low) to 500mM(high)Chelating agent (EDTA or EGTA)DetergentProtease inhibitorPhosphatase inhibitor (optional)
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Lysing Cells
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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
SDSNP-40Triton X-100Tween 20DigitoninCHAPSBrig
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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
AprotininLeupeptinPMSF (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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Phosphatase inhibitors
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Need to add to lysis buffer if using phopshospecific antibodies or suspect protein of interest is phosphorylated
InhibitorsZnCl2
NaFNa-Vanadate (tyrosine phosphatase inhibitor, add prior
to lysis since only active in pH 7 for minutes)
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Further denature proteins
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Add lysate either a known concentration of proteins or cell number equivalent to SDS loading buffer
SDS Loading BufferBuffer (Tris-Cl pH 6.0)2% SDS0.1% bromophenol blue10% glycerol (allows sol’n to sink to bottom of gel wells)-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 lysateAsheesh Pandey
Ingredients in Gel
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Sodium dodecyl sulfate (SDS)Tris buffer (either glycine or tricine)Acrylamide and NN-bis-acrylamide
Forms gel matrixTEMED
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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Pouring your gel
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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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Pouring gel continues
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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 porosityDeposits 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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Loading gel
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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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Running Gel
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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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Considerations with PAGE
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Preparing and Pouring GelsDetermine pore size
Adjust total percentage of acrylamideVary amount of crosslinker
Remove oxygen from mixtureInitiate polymerization
Chemical methodPhotochemical method
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Considerations with PAGE
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Analysis of GelStaining 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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SDS Gel Electrophoresis
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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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SDS Gel Electrophoresis
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Continuous and Discontinuous Buffer Systems
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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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Continuous and Discontinuous Buffer Systems
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Commonly used protein stains
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Stain Detection limit
Ponceau S 1-2 g
Amido Black 1-2 g
Coomassie Blue 1.5 g
India Ink 100 ng
Silver stain 10 ng
Colloidal gold 3 ng
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Chromogenic stains
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Fluorescent stains
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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
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Blotting
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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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Isoelectric PointThere 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.
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Isoelectric Focusing
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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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Two-Dimensional Gel Electrophoresis
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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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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).
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O’Farrell 2D Gel System
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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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• 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
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• 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.
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• 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
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BLOTTING
1. SOUTHERN BLOT
2. NORTHERN BLOT
3. WESTERN BLOT
Blotting: History
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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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SOUTHERN BLOTTING ?
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Experimental procedure DNA is extracted from cells, leukocytes.
DNA is cleaved into many fragments by �restriction enzyme (BamH1, EcoR1 etc)
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VNTR
STR
Microsatelite
Macrosatelite
RFLP
AFLP
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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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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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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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Southern blot analysis depend on the specific restriction endonuclease
The probe used to visualize the restriction fragments.
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•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. 32P
•Sensitive•Relatively cheap•HazardousYou should follow the radioactive waste disposal regulations.
•Sensitive•Relatively expensive
Target DNA
ProbeBiotin Avidin
*
Target DNA
Probe*
Probes
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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
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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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StepsDigestion 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)
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METHODS OF TRANSFER
Downward Capillary Transfer
Upward Capillary Transfer
Simultaneous Transfer to Two Membranes
Electrophoretic Transfer
Vacuum Transfer
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Example of TransferUpward Capillary Transfer
WeightGlass Plate
Whatman 3MM paper
Gel
Paper towels
Membrane (nylon or nitrocellulose)
Whatman 3MM paper
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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
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Northern BlottingNorthern 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
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Basis Steps of NB1. Isolation of intact mRNA2. Separation of RNA according to size (through a
denaturing agarose gel e.g. with Glyoxal/formamide)Transfer of the RNA to a solid supportFixation of the RNA to the solid matrixHybridization of the immobilized RNA to probes complementary to the sequences of interestRemoval of probe molecules that are nonspecifically bound to the solid matrixDetection, capture, & analysis of an image of the specifically bound probe molecules.
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ApplicationsStudy 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
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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
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Western Blotting“Immunoblotting”= electrophoretic transfer of proteins from gels to membranes
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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 proteins Towbin H, et al (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and from 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): 4350–4354 76 (9): 4350–4354
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Applications & AdvantagesApplications:
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 techniqueAs little as 1-5 ng of an average-sized protein can be detected by WB
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Western blotting
The main steps of blotting technique in a chronological order will be as follows:
BlockingProbing with the specific antibody(ies)WashDetection WashingX-ray (Gel Documentation System)
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Electrophoretic Transfer: An Overview
Important Issue:Important Issue:Where to put the gel and the membrane relative to the Where to put the gel and the membrane relative to the
electroblotting transfer electrodes?electroblotting transfer electrodes?75 Asheesh Pandey
Direction of Transfer
Perpendicularly from the direction of travel of proteins through the separating gel
Gel
Membrane
Probe with specific Ab
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Factors Affecting Transfer Efficiency1. The Composition of the gel2. Whether there is complete contact of the
gel with the membrane3. The position of the electrodes4. The transfer time5. The size & composition of proteins6. The field strength7. The presence of detergents
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WB Procedure; Briefly…12
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Direct Detection Method
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Indirect Detection Method
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WB: examples of used substrates
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Chemiluminescent substrates
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Enhanced ChemiFluoresenct (ECF) WB Detection
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Western Blotting Procedure; Illustrated
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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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Steps of WB
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For Direct Transfer, choices are:
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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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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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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.
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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.
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Comparison between WB & SB.
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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).
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References
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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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