Separation Methods: Electrophoresis/Electro-Blotting

Define each type of blotting!

(i) Southern Blotting involves the transfer of DNA from a PAGE or agarose gel to a blotting membrane (nitrocellulose, nylon or polyvinylidene fluoride) and identified using DNA or RNA probes! (ii) Northern Blotting refers to the transfer of RNA to a blotting membrane (diazobenyzloxymethyl groups (covalent binding) or nitrocellulose (binds RNA poorly)) and identified using DNA or RNA probes! (iii) Western Blotting refers to the transfer of proteins from a gel to the blotting membrane (nitrocellulose or polyvinylidene fluoride) and identified typically using an antibody!

Native gel electrophoresis is run in the ________ of sodium dodecyl sulfate (SDS).

Absence

How does Cellulose Acetate Electrophoresis work?

Cellulose Acetate Electrophoresis separate proteins primarily by charge. • Cellulose acetate is a plastic material that can be made in the form of thin strips. • The strip has pores and channels so that ions can migrate through their structures. • The control of pH is very important a buffer solution of the proper pH is used instead of just water. The buffer in the anode and cathode compartments is the same as the buffer in the center of the tank. • The strip of cellulose acetate are soaked in the buffer before use and then are mounted in a cell. •The sample (e.g. serum protein) is applied to the presoaked strip with an applicator. When serum is placed on a cellulose acetate strip, electrophoresis is performed. •The proteins will be separated into several fractions. •If the proteins are stained and the white background of the strip is cleared to colorless, the strip would look something like this figure.

What is electro-blotting?

Electro-Blotting is a technique employed to transfer DNA, RNA or Proteins from an electrophoretic gel (gel volume) to the surface of a membrane (blotting membrane).

What is a major problem with cellulose acetate electrophoresis?

Heat buildup (can be a big problem) ---• If the strip gets too hot, buffer solution will evaporate off the strip or the sample may be destroyed. The current is kept low ~~> the buffer may be refrigerated before or during use. The heating limitation imposed on current restricts the applied voltage ~~> the lower voltage, the slower the separation will be.

Define Hybridization

Hybridization : process where two complementary single strands of nucleic acid (DNA or RNA) form a double helix.

What is Northern Blot mainly used for?

Northern blots are mainly used to measure of the amount of a specific RNA in the cell!

Blotting Apparatus Side Note

Note, buffer pH is adjusted to affect an analyte net charge appropriately, relative to positioning of the anode and cathode. High amperage is required (0.1 - 1 amp)

How does Native Gel Electrophoresis work?

Separation by size and charge (charge/mass)!!!

What are the types of Vertical Gels?

Slab gels : uses flat gels formed between two plates of glass - Allow for two dimensional analysis. - Running multiple samples simultaneously in the same gel. • Tube gels: column electrophoresis, uses tubular gels formed in glass tubes. - a slightly improved resolution of the bands - more economical (relatively easy to construct homemade systems)

3 Main Hybridization Techniques

Southern Blotting Northern Blotting Western Blotting

What are the different ways of analyzing mixtures/analytes/ions, ect.?

Spectrochemical Methods Electrochemical Methods Separation Methods Nuclear Chemistry Methods

What does the blotting process do to the analyte?

The blotting process CONCENTRATES the analyte on a surface for FURTHER ANALYSIS.

What does western blot refer to?

Western Blotting refers to the transfer of proteins from a gel to the blotting membrane (nitrocellulose or polyvinylidene fluoride) and identified typically using an antibody.

Electroendosmosis

With a pH 8.0-9.0 used for protein electrophoresis, proteins take on a negative charge, that is a negative ion cloud forms ~~> migrate to the anode. As the negative ion (protein) cloud migrates to the anode, the proteins are pulled to the anode. Several supporting substrates used routinely for protein electrophoresis attract positive ions from the buffer and form a positive ion cloud. This ion cloud moves in the opposite direction to the cathode. This phenomenon is called Electroendosmosis or Endosmosis these oppositely moving ion clouds can affect the movement of sample macromolecules. The migration of some proteins can be slowed, some proteins can become immobile, and other proteins are pushed toward the cathode. Many protein electrophoresis methods take advantage of this tension and use it to achieve better separation of protein bands A nonionic, nonpolar compound is used (not move by itself under an electric field).

What materials does gel electrophoresis use?

involves the use of a gelatinous material such as agarose,cellulose acetate, acrylamide, or starch as the matrix. The gel acts as a support medium for the sample.

In native polyacrylamide gel electrophoresis (PAGE) the mobility depends on what?

the mobility depends on both the protein's charge and its hydrodynamic size!!!

What is Electrophoresis?

• A technique whereby charged molecules are separated by the use of an electric field.

Agarose vs Acrylamide

• Agarose is a polysaccharide similar to starch useful in separating DNA and RNA molecules of a few hundred to tens of thousands on bases long. • Poly Acrylamide (PAGE) is a cross-linked synthetic polymer useful in separating DNA molecules of a few hundred bases in length.

Western Blot Details

• Extraction of protein from transformed cells. • Separation of protein by using SDS-PAGE where SDS acts as solvent for electrophoresis. • The blot is prepared and hybridized like a southern blot. • Blotting of proteins into nitrocellulose filter paper. • Placing of whatmann filter paper on a cathode plate followed by stack of coarse filter, whatmann filter, electrophoresed gel, nitrocellouse filter, coarse filter stack, whatmann filter and anode plate. • Putting the complete set up in transfer tank containing sufficient transfer buffer. • Application of an electric field cause the migration of proteins from gel to nitrocellulose filter.

Types of Separation

• Native: separations are run in non-denaturing condition by size and charge (charge/mass) • Denaturing: Proteins or nucleic acids lose the tertiary structure and secondary structure. separation by size (The larger molecules move slowly thorough gel) • Others (Isoelectric focusing (IEF), 2-Dimesional gel electrophoresis)

What are the main factors that effect separation?!

• Resistance (pore size)! • Buffer strength! • Gel Temperature! • Sample!

Types of Electrophoresis

• Supporting medium electrophoresis (chromatography paper, film, various gels) ~~~> The sample migrates through a supporting material that minimizes the amount of diffusion. • Free solution electrophoresis (Capillary Electrophoresis)

Why do proteins move through the gel at different rates?

• The relative rate of movement depends upon the molecular weight, shape and charge of that protein. SDS-treated proteins are subjected to electrophoresis the rate of migration is determined ONLY by the molecular weight of the protein.

What type of technique is SDS PAGE?

•Sodium Dodecyl Sulfate - Polycrylamide Gel Electrophoresis (SDS-PAGE) is an important protein electrophoretic technique. - SDS-PAGE allows for protein to be separated on the basis of molecular weight and not charge!!!

Northern Blot Details

•The blot is prepared and hybridized like a southern blot • There are a few minor technical alterations - The RNA is usually folded into a secondary structure so it must be denatured before and during electrophoresis • Bands on the autoradiogram are a measure of the presence of RNA from a particular gene - The more intense the band, the more RNA on the blot