There are various techniques for the separation and characterization of proteins. They include chromatography and electrophoresis. The choice of technique for a particular mixture requires knowledge of the protein molecules of the mixture.

A mixture of fluids can be easily separated from one another due to differences in solubility in different solvents and if the constituents of the mixture have differing molecule sizes.

However, for proteins that have similar physical and chemical properties, then chromatography has to be the technique employed to purify the mixture. Electrophoresis is a major technique for laboratory protein separation because it is relatively easy and cheap to carry out.

Available Techniques Involving Chromatography

Chromatography works by putting the substances to be separated into a structure that consists of two phases, that is: a mobile phase and a stationary phase. These substances will then be separated based on differences in their interaction between these two phases as the mobile phase moves across the stationary phase.

In the liquid chromatography technique, the substance (solute) to be analyzed is dissolved in the solvent. The solvent then flows across a solid matrix (which is the stationary phase).

The solutes then intermingle with the stationary phase by reversibly binding to the stationary phase. The potency of the binding between the solute and the stationary phase will determine how fast the solute is carried by the mobile phase.

1. Column Chromatography

Column chromatography consists of a central component known as the column. A pump is included to control the rate of flow of buffers through the column. The solvent used in the mobile phase will need to gradually change and this is with the aid of a gradient maker.

The elution of the different components of the solute can be noted during chromatography either with a spectrophotometer which measures the absorbance of material coming off the column or for protein chromatography, monitor the A280.

Chromatography which is used for protein purification schemes will include a fraction collector. A fraction collector is an apparatus that routinely collects the liquid flowing from the column into separate tubes.

Each tubes are then evaluated for the proteins investigated. The quantities of the proteins will be plotted against either the fraction number, volume, or time. These three variables can be easily determined from the flow rate and volume of the individual fractions.

2. High Performance (Pressure) Liquid Chromatography

High Performance (Pressure) Liquid Chromatography is an improvement in technology over column chromatography. The flow rates in column chromatography are restricted due to the compression of the support matrices present in the columns.

This technology employs the use of resins that can endure the rigors of packing and high flow rates such that high resolution is enhanced.

Another advantage is that the separation of substances can be done under higher pressures such as high flow rates which will result in increased resolution.

3. Adsorption Chromatography

The thin-layer chromatography (TLC) and paper chromatography are typical examples of adsorption chromatography. Adsorption chromatography is normally utilized for the separation of small molecules e.g. lipids, nucleotides, simple sugars, and amino acids.

Although adsorption chromatography is not commonly used in protein purification, hydroxyapatite columns have been utilized successfully in exceptional situations to separate proteins that were otherwise not easily separated by other techniques.

Hydroxyapatite is a crystalline form of calcium phosphate. It has been used as a stationary phase medium to analyze proteins or nucleic acids. It involves a non-specific interaction between the positive calcium and negative carboxyl groups on the proteins and the negative phosphate and positive amino groups on the protein.

4. Ion Exchange Chromatography

Ion exchange chromatography (IEC) is a variant of adsorption chromatography. It is based on charge-charge interactions. The stationary phase includes fixed charges on a solid support.

The fixed charges on the stationary phase are either positive or negative. These charges are referred to to as anion exchange or cation exchange chromatography respectively.

The mode of action is such that the substances that are to be separated will replace the counter ions associated with the chromatography medium and firmly bind to the exchanger by way of electrostatic interactions.

In the situation that some solute molecules are electrostatically bound to the exchanger and other solute molecules are not bound is used to separate solutes (proteins).

A typical example is the mixtures of adenine nucleotides, consisting of adenosine, adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) can be separated by this method.

Adenosine is uncharged and hence will not bind to the anion exchanger. With an increment in the concentration of formate to the column, AMP will be the first to be eluted, next is ADP, and lastly the ATP.

The sequence of elution is related to the overall negative charge of the nucleotides (i.e. the number of phosphate groups).

5. Hydrophobic Chromatography

Hydrophobicity is a chemical property that causes the aggregation of nonpolar compounds with each other in an aqueous environment. Proteins can be separated according to differences in their hydrophobicities.

The media for hydrophobic chromatography is a support matrix made of agarose and long-chain hydrocarbons covalently bound to it. Examples include octyl agarose (8 contiguous methyl groups) and phenyl-agarose.

They (octyl-agarose and phenyl-agarose) provide a hydrophobic surface for proteins to interact with instead of aggregating with each other. Highly hydrophobic resins such as octyl-agarose are most suitable for weakly hydrophobic proteins, while less hydrophobic resins such as phenyl-agarose are more suited for proteins of intermediate hydrophobicity.

Proteins can bind to hydrophobic columns when the conditions that will promote hydrophobic interactions exist as these conditions will determine the extent of binding. E.g. an increase in ionic strength increases hydrophobic interactions. Ammonium sulfate (NH4)2SO4 and sodium chloride NaCl are examples of salts that are used in hydrophobic chromatography.

6. Gel Filtration Chromatography

This technique is also known as molecular sieve chromatography or size exclusion chromatography. The technique separates proteins based on individual molecule size. The protocol is such that the test substance (proteins) is passed over a column made of small beads (composed of cross-linked polymers).

The pore size is determined by the extent of cross-linking of the polymers. Proteins (solutes) which.are smaller than the pore easily go into the gel matrix and are retained on the column for a longer duration.

However, protein (solute) molecules that are bigger than the pore size will not enter the matrix or beads but pass through the column unhindered. The duration of retention of solute in the gel matrix is inversely proportional to the size of the molecule of the protein (solute).

Gel filtration has been used to determine the molecular weight of a protein. This is possible if the columns are calibrated by using markers (proteins) of known molecular weight.

These markers whose molecular weights are known are passed over the column and the Kav is determined for each protein as follows:

Kav = Ve – Vo/Vt – Vo.

Where;

Vo = void volume (It is the volume of the substance that is too large to enter the matrix of the support medium.

Vt = total volume (is calculated from the volume of the column bed i.e πr2 x length)

Ve = elution volume

7. Affinity Chromatography

Affinity chromatography involves protein interactions with specific ligands. The protocol is for the protein mixture to be passed over the column with a suitable specific segment of the molecule which is attached by a covalent bond to a solid support.

The protein which has affinity to the ligand of interest will be the only protein to bind to the column while the other proteins will be washed away. Affinity chromatography is very precise and enables the isolation of a single protein in a single step.

Techniques Involved In Separation and Characterization of Components Involving the Use of Chromatography and Electrophoresis

Separation of Proteins Using Electrophoresis

The separation of proteins using electrophoresis is dependent on the charge distribution of the protein molecules that are being separated. Electrophoresis of proteins is usually carried out in solution as the capacity to split these molecules is dependent on their ability to diffuse.

Polyacrylamide gels or agarose gels are good media for achieving great resolutions. However, polyacrylamide is the choice gel for protein electrophoresis while agarose gel is more commonly used for nucleic acids.

This is because agarose gels have larger pore sizes than acrylamide gels thus more effective for bigger macromolecules. Both types of gels may however be used for either proteins or nucleic acids.

1. Polyacrylamide Gel

Sodium dodecyl sulfate (SDS) is a detergent that can be added to proteins. The proteins become negatively charged when they interact with the SDS anions.

SDS will affect the protein-protein interaction thus denaturing virtually all proteins present in the test substance which will reveal the entire proteins present.

A situation whereby these proteins are separated on a polyacrylamide gel is referred to as Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis or SDS–PAGE.

It is the protocol used for the determination of molecular weight. The following is a hypothetical procedure for SDS-PAGE.

Materials required:

1. SDS-polyacrylamide gel

2. Protein markers

3. 2X-SDS Sample Buffer

4. 1X-SDS Electrophoresis Running Buffer (i.e. Tris-Glycine + SDS)

5. 0.001% (w/v) Bromophenol Blue

6. Micropipettes with disposable tips

7. 0.25% (w/v) Coomassie Brilliant Blue R 250 in methanol-water-glacial acetic acid (5-5- 1), filtered immediately before use.

8. 7% (v/v) acetic acid

Procedure;

1. Pour the separating gel which should contain about 6-20 percent acrylamide. Note that the choice of the acrylamide concentration is dependent on the weight of proteins being separated, the desired resolution, and the quantity of sample

2. Pour stacking gel after the separating gel has polymerized and just before electrophoresis.

3. This will minimize diffusion between the two gels.

4. Load test samples. The proteins to be analyzed should first be solubilized in a sample buffer (containing 2% SDS and 5% β-mercaptoethanol) and then boiled.

5. Load the [protein marker on the first and last wells of the gel. Avoid overloading the wells as this results in the pores in the results of the gel becoming plugged. A tracking dye (bromophenol blue) should also be included in each sample.

6. The function of the dye is that when it reaches the bottom of the separating gel, the power can be turned off and the proteins detected

7. An electric field is applied after loading the samples into the wells of the gel to run across the gel (20 mA constant current per 1.5 mm gel). If excessive heat is applied, this may result in the proteins running off the gel.

8. Stain or process gel. To detect proteins after electrophoresis, the gel is processed by staining with Coomassie blue (a dye that binds proteins).

9. The gels are then ‘fixed’ before staining with an acetic acid and methanol solution which precipitates proteins into the acrylamide matrix.

10. Use at least 10 volumes of Coomassie Blue staining solution for 2-4 hours, tilting gently to aid the distribution of the dye evenly over the gel.

11. Destaining and washing of the gel. After the staining, Wash the gels while changing the water until the blue coloration stops.

12. Place the gel in a 7% acetic acid solution for at least 1 hour and move the gel into fresh 7% acetic acid as required (until the blue background is no longer visible). Place the gel into a container and cover the gel with 7% acetic acid fix proteins. Photograph the gels.

2. Agarose Gel

Agarose gels are formed by heating the appropriate concentration of agarose in an aqueous buffer. When the agarose has dissolved, the solution is allowed to cool down to enable the gel to form. The protein samples are then loaded into the wells while the protein markers are put in the wells on the first and last wells.

In conclusion, despite the many techniques for the separation and characterization of proteins, electrophoresis and chromatography are the techniques of choice.

Knowledge of the physical (molecule size) and chemical properties determines the technique of choice for the separation of proteins into the different components. The choice of technique for separation of protein requires knowledge of the physical and chemical properties of protein molecules in the mixture.

Proteins with similar physical and chemical properties can be separated using the chromatography technique to purify the mixture. Electrophoresis is a major technique for laboratory protein separation as it is very easy and cheap to carry out.