(analytical Biochemistry, Arie Admon, Biology, Technion)

Books: Electrophoresis in practice. R. Westermeier (1993) VCH

Introduction (chapter 0)

Charge, size, shape and friction influence movement of biomolecules in electric field in solution.

Field strength is influenced by distance between electrodes, voltage and conductivity of the buffer.

The pH and salt content may change in during the run. Electro-osmosis may happen.

Samples are proteins, peptides, amino acids, nucleic acid and sugars.

Fig 1.

1. Electrophoresis: field and buffer are constant.
2. Isotachophoresis: field and buffer are discontinuous. Stacking is usually the goal.
3. Isoelectric focusing: The proteins move to their isoelectric point.

Free solution electrophoresis

U-shape tubes are not in use (fig. 2)

Continuos free flow zone is hardly in use (Fig 3). Used for the separation of cells or organelles.

Capillary electrophoresis is very much in use and its use is growing (fig. 4). Capillaries can be used for electrophoresis, isotachophoresis and for isoelectric focusing. Capillaries can be bare silica or coated with acrylamide or methyl cellulose or filled with gel or viscous buffer. Electro-osmotic flow is either a problem or an advantage. Auto sampling is fraction collection are possible. Detection with UV or fluorescence.

Electrophoresis in supporting media.

Detection can be done during (fluorescence) or after the run (UV, fluorescence, blotting, staining, auto-radiography, precipitation, immuno-precipitation and enzymatic activity.

Paper and thin layer in cellulose or silica gel.

Cellulose acetate electrophoresis hardly used at all.

Gel electrophoresis is the most common method. It is performed in tubes, vertical slab gel and horizontal gel on support film (fig 5).

Agarose gel with pore size from 150 nm (1%) to 500 nm (0.16%) (fig 6). It is mostly used in flat gel as its mechanical strength is not good.

Polyacrylamide (fig 7) is used in flat, slab and capillary. Pore size is controlled by total acrylamide (T) and cross linking (C )

T=(a+b)x100x[%]/V

C=bx100[%]/(a+b)

a= acrylamide in gram

b= methylbisacrylamide in gram

V= volume in ml

Remember: monomers are toxic. Oxygen prevents polymerization as a free radical trap, so oxygen diffusion should be limited. Temperature should be above 20 C.

Electrophoresis in non-restrictive gels and detection by immuno-fixation.

Electrophoresis in restrictive gels.

Ferguson plot: The log ₁₀ of M_r versus the gel concentration gives a straight line. The slope depends on the protein.

Agarose gel is mostly used for DNA and RNA. Acrylamide for all samples. DNA sequencing in slab and in capillary. Heat and urea for denaturation.

Proteins in native and in SDS-PAGE.

Linear and gradient gels (fig 17 and fig 19).

Sample treatment Reducing the S-S bonds and protecting the SH groups with iodoacetamide, iodoacetic acid and vinylpridine. Denaturing with heat, SDS (and urea).

SDS is a good denaturant and gives a high and uniform charge density. After removal of SDS the proteins are fixed.

Phosphate, tris-glycine, tricine and tris-acetate can be used as buffers.

Isoelectric focusing (chapter 3)

The charge of the protein depends on the pH. Post-translational modifications affect the net charge.

Movement to the isoelectric point depends on the charge and shape. Given enough time all proteins will reach the isoelectric point (fig 24) therefore sharp bands can be obtained. Very flat pH gradients can be used. Free flow through membranes can be used.

Carrier ampholytes form a pH gradient during pre-focusing (fig 25). A pH range of 3-10 can be obtained. Plateau effect in the middle and drift of ends.

Slab gel isoelectric focusing is very useful for comparisons (fig 26).

Immobilized pH gradients: The Immobilines are co polymerized into the acrylamide gel (fig 27). The gel is polymerized as in gradient gel with a linear gradient of Immobilines.

Addition of urea, non-ionic or zwitterionic detergents. Temperature should be kept low enough to prevent changes in the protein but high enough to prevent crystallization of the urea.

Two dimensional electrophoresis

Increased use of isoelectric focusing in fist dimension and SDS-PAGE in the second (2D gel). First dimension of isoelectric focusing or immuno or native gel.

First dimension in tubes, stripes, slab and agarose.

To prevent drift during run, an immobilized pH gradient is used (fig 21). This is the highest resolving power technology currently available for proteins.

Proteome analysis is becoming fashionable.

Blotting (chapter 4, fig 31)

Blotting after gel electrophoresis for sequencing, hybridization, western.

Diffusion blotting, capillary blotting and vacuum blotting. Electrophoretic blotting in tank and semi-dry.

Film supported gels preparation for blotting (fig 36).

Blotting membranes: Nitrocellulose is very good except that it dissolves in organic solvents. PVDF is stronger mechanically and does not dissolve so it can be used for direct sequencing. Nylon is mostly used for DNA RNA. Ion-exchange membranes for preparative work. Activated glass fiber for direct sequencing.

Staining

Silver

Coomassie

Colloidal

Fluorescent stains