BioOutsource

Protein Characterisation

SDS-PAGE (reducing/non-reducing/native)

Protein separation by SDS-PAGE (polyacrylamide gel electrophoresis) can be used to estimate relative molecular mass (molecular weight) and determine the relative abundance of major proteins in a sample and to determine the distribution of proteins among fractions. 

The test protein is prepared in solution with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. The SDS binds to and linearises the protein giving an approximately uniform mass to charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.  Besides the addition of SDS, proteins may also be heated to near boiling in the presence of a reducing agent (such as DTT), which further denatures the proteins by reducing disulfide linkages, breaking up quaternary protein structure.  This is known as reducing SDS-PAGE, and is most commonly used. Non-reducing SDS-PAGE (no boiling and no reducing agent) may be used in situations when native structure is important in further analysis.

Isoelectric focusing (IEF)

Isoelectric focusing is a technique for separating different molecules by their charge differences.  It is a type of zone electrophoresis, usually performed in a polyacrylamide gel, that takes advantage of the fact that a molecule's charge changes with the pH of its surroundings.

Molecules to be focused are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes).  An electric current is passed through the medium, creating a "positive" anode and "negative" cathode end. Negatively charged molecules migrate through the pH gradient in the medium toward the "positive" end while positively charged molecules move toward the "negative" end.  When the applied proteins reach the gel fraction that has a pH that is the same as their isoelectric point (pI), their charge is neutralised and migration ceases.  The pH gradient is initially established before adding the particles of interest by first subjecting a solution of small molecules such as polyampholytes with varying pl values to electrophoresis.

Gels with large pores are used in this process to eliminate any "sieving" effects, or artefacts in the pI caused by differing migration rates for proteins of differing sizes. Isoelectric focusing can be used as an identity test when the migration pattern on the gel is compared to a suitable standard preparation and IEF calibration proteins.  It can also be used as a limit test when the density of a band is compared subjectively with the density of bands appearing in a standard preparation, or it can be used as a quantitative test when the density is measured using a densitometer to determine the relative concentration of protein in the bands.  IEF can resolve proteins that differ in pl value by as little as 0.01.

Electrophoretic (Western) blotting

An analytical technique in which prior to protein immobilization on the PVDF or nitrocellulose membranes, sample proteins are separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE) before detection using monoclonal or polyclonal antibodies.  This technique can provide information about molecular weight and the potential existence of different isoforms of the proteins under study.

pH, appearance and osmolality

pH, appearance and osmolality are general tests required for final product testing.

The assessment of particles in solution is determined by visual inspection. The pH (potential of hydrogen) is determined using a validated Mettler Toledo SevenEasy™ S20K pH meter and InLab® Expert Pro combination electrode, checked for accuracy using standards. Osmolality is measured using the Osmomat® 030, which is calibrated by performing measurements with distilled water and two calibration standards.

Osmolality (osmoles per kg solution) is a measure of the total concentration of dissolved particles in a solution, without regard for homogeneity of the molecular species or the molecular weights, size or density. Any substance dissolved in a solvent affects four colligative properties of the solvent-solute mixture: decrease in vapour pressure, decrease in freezing point, increase in boiling point and change in osmotic concentration. All of these properties are interrelated and the accurate determination of any one of these properties allows the estimation of the other three and is a measure of the osmotic concentration of the solution.

Freezing point depression is one of the easiest methods for determination of the osmotic concentration of biological fluids. The sample is placed in a cooling chamber maintained below the freezing point of the solution, and then supercooled which initiates crystallisation. Crystal formation results in the release of heat, causing the sample to warm to the point at which ice and solution exist in equilibrium.

Osmotic concentration (osmolality) is expressed in units of milliOsmoles (mOsm) per kg of water where one Osm is equivalent to one mM of dissolved solute particles. A solution containing one Osmole (1000 mOsm) of dissolved solute per kg of water lowers the freezing point of water by 1.858oC. Therefore, the freezing point depression of the sample can be converted to units of osmolality by dividing by 1.858. This calculation is performed automatically by the osmometer.