Electrophoresis

clinical chemistry

1. ELECTROPHORESIS BASIC CONCEPTS, INSTRUMENTATION, TECHNIQUE, TYPES AND APPLICATIONS By Dr . Basil, B – MBBS (Nigeria) , Department of Chemical Pathology/Metabolic Medicine, Benue State University Teaching Hospital, Makurdi . February, 2015.

2. INTRODUCTION ELECTROPHORESIS – migration of charged solutes or particles in a liquid medium under the influence of an electric field The aim of carrying out electrophoresis include: To determine the number, amount and mobility of components in a given sample or to separate them. To obtain information about the electrical double layers surrounding the particles. Determination of molecular weight of proteins and DNA sequencing.

3. BASIC CONCEPTS Iontophoresis – migration of small ions Ionized species move towards the anode or cathode depending on their charges Ampholytes become + vely charged in a soln more acidic than its isoelectric point ( pI ), and – vely charged in a more alkaline soln Rate of migration depends on: Net electrical charge of the molecule Size and shape of the molecule Electrical field strength Properties of the supporting medium Temperature of operation

4. Electrophoretic mobility (µ) – rate of migration (cm/s) per unit field strength (volts/cm) µ = Q/6∏rȠ Where: µ = electrophoretic mobility in cm2/(V)(s) Q = net charge on the ion r = ionic radius of the solute Ƞ = viscosity of the buffer soln Other factors affecting mobility: Endosmotic flow: This is the preferential movement of water in one direction through an electrophoresis medium due to selective binding of one type of charge on the surface of the medium Wick flow: Movement of water from the buffer reservoir towards the center of an electrophoresis gel or strip to replace water lost by evaporation

5. INSTRUMENTATION/REAGENTS

6. Power Supply: Electrophoresis is done under conditions of constant voltage, current or power. Current (I) flow through the medium > heat production due to resistance (R) > increase in thermal agitation of dissolved ions > decrease in resistance and increase in current flow >> more heat and evaporation of water from the buffer > increase in ionic concentration of the buffer Migration rate is kept constant by using a constant-current power supply. Since, EMF = IR, and increase in R will result in and increase in EMF (V) at constant current (I), thus, no appreciable heat production

7. Buffers: F unction include: Carries the applied current Establishes the pH of the system Determines the electrical charge on the solute The ionic strength influences: Conductance of the support Thickness of the ionic cloud (buffer and non-buffer ions) surrounding a charged molecule Rate of its migration Sharpness of the electrophoretic zone Features of an ideal buffer: Does not interfere with the ability to detect the analytes of interest Maintains solubility of the analytes Maintains buffering capacity through the analysis Produce the desired separation

8. Commonly used buffers include: Low pH (acidic): phosphate, acetate, formate , citrate High pH (basic): tris , tricine , borate, CAPS (N-cyclohexyl-3-aminopropanesulfonic acid ) Higher the ionic strength (and concentration) > higher size of ionic cloud > lower mobility of the particle Also, higher ionic strength > sharper protein-band separation and increased heat production > denaturation of heat-labile proteins Buffers used are made of monovalent ions because their valencies (ionic strength) and molality are equal. They are good culture media for microorganisms and should be refrigerated when not in use, Also cold buffer reduces evaporation and improves resolution Small volumes should be discarded after use, but large volumes can be reused up to 4 times

9. Support Media: Insoluble gels e.g sheets, slabs or columns of starch, agarose or polyacrylamide Membranes (paper) of cellulose acetate Pure buffer soln in a capillary Starch Gel: Separate macromolecules on basis of surface charge and molecular size Obsolete because preparation of reproducible starch gel is difficult Cellulose Acetate: made by treating cellulose with acetic anhydride They need to be soaked in buffer to soften them before use Require clearing before densitometry Hardly used in routine clinical application

10. Agarose: Used in AGE for the separation of Serum, Urine or CSF proteins, Hb variants, Isoenzymes , Lipoproteins etc. Separation is based only on charge-to-mass ratio – pore size is large enough for all proteins to pass Separate proteins into only 5 fractions (zones): albumin, alpha-1, alpha-2, beta- and gamma-globulins Advantages: Permits excellent densitometry – lower affinity for proteins (migration is not affected) and native clarity after drying Little Endosmosis – free of ionizable groups (neutral) Disadvantage: DNA recovery is affected by inhibitors

11. Polyacrylamide: thermostable, transparent, durable and relatively chemically inert No endosmosis – uncharged Pore size does not allow larger proteins like fibrinogen, B1-lipoprotiens, Y-globulins etc , to migrate Separation is based on both charge-to-mass ratio and molecular mass – molecular sieving Carcinogenic – CAUTION when handling Accommodates a large amount of sample in a single sample slot DNA recovered is pure with no inhibitors unlike Agarose. Separates proteins into 20 or more fractions Used to study individual proteins ( e.g Isoenzymes )

12. Equipment used for Gel Electrophoresis:

13. TECHNIQUE Separation: Blot the hydrated support medium to remove excess buffer The sample is added to the support for about 5min The support is then placed into the electrophoretic chamber in contact with the buffer Apply a constant-current or constant-voltage power for a specified time The support is then removed and placed in a fixative or rapidly dried to prevent diffusion of the sample

14. Staining: The support is then stained and dried after washing out the excess dye Amido Black B or members of the Coomassie Brillant Blue series are the commonest dyes The amount of dye taken up is dependent on the type of protein, degree of denaturation by the fixing agent and quality of the dye To visualize isoenzymes , incubate the gel in contact with a solution of substrate, which is linked structurally or chemically to a dye, before fixing. Typical stain solution may be used several times When the stain solution becomes faulty, protein zones will appear too lightly stained It must be stored tightly covered to prevent evaporation

15. Detection and Quantification: Detection can be achieved using UV light, but quantification is by Densitometry Densitometers integrate the area under a peak, and the result is printed as percentage of the total Reliable quantification of stained zones using densitometry requires: Light of an appropriate wavelength Linear response from the instrument Transparent background in the strip being scanned Mass spectrometers – determine the molecular weights of proteins and their cleavage products, and for peptide sequencing.

16. Blotting Techniques: Southern Blotting – widely used in molecular biology: DNA or DNA fragments separated by AGE Strip of nitrocellulose or a nylon membrane is laid over the gel blotting the DNA or DNA fragments onto it by capillary, electro-, or vacuum blotting Detection and identification is by hybridization with a labeled complementary nucleic acid probe. Northern Blotting: Separates and detects RNAs Uses labeled RNA probe for hybridization Western Blotting: Separates and detects proteins The membrane is reacted with a reagent containing an antibody raised against the protein of interest

17. Common Problems Encountered:

18. TYPES OF ELECTROPHORESIS Zone Electrophoresis Slab Gel Electrophoresis Disc Electrophoresis Isoelectric Focusing Electrophoresis 2-Dimensional Electrophoresis Capillary Electrophoresis Microchip Electrophoresis

19. Zone Electrophoresis: Produce zones of proteins that are heterogeneous and physically separated from one another Classified according to type and structure of the support material e.g AGE, CAE, PAGE etc

20. Slab Gel Electrophoresis: Use of a rectangular gel regardless of the thickness Main advantage – ability to simultaneously separate several samples in one run Primary method used in clinical chemistry lab Gels (usually agarose) may be cast on sheet of plastic backing or completely encased within a plastic walled cell allowing horizontal or vertical electrophoresis and submersion for cooling, if needed. May be cast with additives like: Ampholytes which create a pH gradient, or Sodium dodecyl sulfate (SDS) that denatures protiens

21. Schematic representation of Slab gel electrophoresis

22. Useful in the separation of serum proteins, isoenzymes , lipoproteins, hemoglobins , and fragments of DNA and RNA Common problems encountered in SGE include: Discontinuities in sample application – dirty applicator; cleaned by agitating in water Unequal migration of samples across the width of the gel – dirty electrodes (uneven application of electric field), uneven wetting of the gel Distorted protein zones – bent applicator, incorporation of air bubble during sample application, over application of sample, excessive drying of support Unusual bands – artefacts, other causes e.g hemolysed samples > increased b-globulin band Atypical bands – denatured protein from deteriorated serum, also rule out a true paraprotein

23. Disc Electrophoresis: 3-gel system: small-pore separating gel ( running gel ), a larger-pore spacer gel ( stacking gel ), and a thin layer of large-pore monomer solution ( sample gel ) – containing about 3 µL of serum The different composition cause disc ontinuities in the electrophoresis matrix During electrophoresis, all proteins migrate easily through the large-pore gels and stack up on the separation gel in a very thin zone This improves resolution and concentrates protein components at the border (or starting zone ) Separation occurs at the bottom separation gel by the molecular sieve phenomenon

24. Schematic representation of Disc Electrophoresis

25. Isoelectric Focusing Electrophoresis: Separates amphoteric compounds with increased resolution in a medium possessing a stable pH gradient The protein migrate to a zone in the medium where the pH of the gel matches its pI At this point, the charge of the protein becomes zero and its migration ceases – it become “ focused ”. Regions associated with a given pH are very narrow – enough to separate proteins that differ in their pI values by only 0.02pH units A high voltage power source is needed because carrier ampholytes are used in relatively high concentrations Thus, the electrophoretic matrix must be cooled IEF is used in neonatal screening programs to test for variant Hb

26. 2-Dimensional Electrophoresis: Uses charge-dependent IEF (first dimension) and m olecular weight-dependent electrophoresis (in the second) 1st dimension – carried out in a large-pore medium like agarose or large-pore PAG; to which ampholytes are added to yield a pH gradient 2nd dimension is often polyacrylamide in a linear or gradient format It achieves the highest resolving power for the separation of DNA fragments 1st dimension – normal AGE 2nd dimension – ethidium bromide is added to the gel to open up the fragments and cause changes in their mobility Method of choice when complex samples need to be arrayed for characterization, as in proteomics.

27. Schematic representation of 2D Electrophoresis

28. Capillary Electrophoresis: Separation in narrow-bore fused silica capillaries (inner diameter 25 – 75µm) filled with buffer – gel media can also be used Sample is loaded after filling capillary with buffer, and electric field applied Borate is a classic CE buffer that generates relatively low current and heat, even with a high ionic strength Electro-osmotic flow (EOF) controls the amount of time solutes remain in the capillary (also Electroendosmostic flow or endosmosis) EOF = bulk flow of liquid towards the cathode upon application of an electric field and it is superimposed on electrophoretic migration Cations migrate fastest due to EOF and electrophoretic attraction towards the cathode

29. Neutral molecules are all carried by EOF but not separated from each other Anions move slowest because EOF is slightly greater than the attraction twds the anode and repulsion from cathode Used for determination of MWts of proteins and peptides, analysis of PCR products, inorganic ions, organic acids, pharmaceuticals, optic isomers, and drugs of abuse in serum and urine . When using a new capillary or changing buffer, the capillary must be equilibrated with the buffer – conditioning Particularly important when phosphate-containing buffer is involved This should be done for at least 4hrs before electrophoresis is started The capillary surface must be regenerated or reconditioned to remove any material adsorbed onto the wall Done by following each run with 3- to 5-column volume rinse using NaOH , and flushing with 5- to 8-column volumes of fresh buffer.

30. Microchip Electrophoresis: Similar in principle to CE, but differs in that the separation channels, sample injection channels and reservoirs are all fabricated into the same planar substrate using photolithographic processes. More so, sample preparationand /or precolumn or postcolumn reactors, detectors, and excitation sources are intergrated into the chip

31. QUALITY CONTROL A Clear SOP must be adhered to during each run to ensure reproducibility Control serum must be included in each electrophoretic run to evaluate its quality and that of the densitometer.

32. APPLICATIONS DNA Analysis: specific DNA sequences can be analyzed, isolated and cloned. The analyzed DNA may be used in forensic investigations and paternity tests . Protein Analysis: In the diagnosis of conditions where levels of specific proteins or total protein is low or higher than normal e.g monoclonal gammopathy etc. Antibiotic Analysis: Synthesis of new antibiotics Analysis of bacteria response to antibiotics and determining antibiotic-resistance Vaccine Analysis: Purification, processing and analysis of vaccines e.g influenza vaccine, hepatitis vaccine, polio vaccine

33. REFERENCES: Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, fifth ed., by Burtis et al. Clinical Chemistry; Principles, Techniques and Correlations, 7th ed., by Bishop et al. http:// www.slideshare.net/jyots23/electrophoresis-presentation http:// www.bio-rad.com/en-us/applications-technologies/protein-electrophoresis-methods Landers JP. Molecular diagnostics on electrophoretic microchips. Anal C hem 2003;75:2919-27 St. claire iii RL. Capillary electrophoresis. Anal Chem 1996;379-423

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