CHAPTER TWO
2.3 Protein sample concentration
2.2.1.2 Procedure
Stock bovine serum albumin (BSA), prepared as in the above, was used to prepare standard curves for the microgram and nanogram range of protein. Five replicates of 40 Ills of the diluted sample were dissolved in 160 Ills of ddH20 each. Each sample (200 Ills) was mixed with 800 Ills of Bradford reagent in 1.5 ml Eppendorf tubes to make up 1 ml. The tubes were gently vortexed and transferred to clean plastic cuvettes. [Only plastic cuvettes were used in the experiment as Coomassie brilliant blue dye binds to glass cuvettes and gives erroneous readings (Bradford, 1976)]. Dye-protein mixtures were allowed to react for two min and absorbance of each sample was measured at 590 run and 450 run within one hour. Double de-ionized water (ddH20) served as a blank instead of dye reagent as in the conventional Bradford's assay because the free dye is also measured at 450 run thus, no free dye control is needed. The ratio ofAS90/4S0 was calculated and a standard curve was plotted against the amount of protein applied. A graph was constructed in Microsoft Office Excel software and a linear regression equation was calculated to determine the concentration of protein.
2.3.1 Salt precipitation
Very dilute proteins for SDS-PAGE analysis may need to be concentrated for detection due to the limited capacity of wells in the gel where protein samples are loaded. Some of the precipitation methods which may be used are the KCl, NaCl, Na2S04 or (NH4)2S04 methods (Englard and Seifter, 1990).
(NH4)2S04 precipitation is important method of protein concentration. In the absence of (NH4)2S04, proteins remain as open structured, more soluble and less stable forms.
Upon addition of increasing (NH4hS04, however, they start to become compact in structure, less soluble and more stable. This occurs as the (NH4)2S04 sequesters more water (the
sol-
binds to 14H20 molecules) and causes dehydration and crowding of the proteins. This eventually causes certain proteins to precipitate as they reach their solubility limit in a process called salting out (Dennison and Loverein, 1997). The use of (NH4)2S04 is chosen because next to citrate, sulphate is the second strongest Hofmeister Cosmothrope which is characterized by its high effectiveness in salting out proteins and stabilizing protein structure (Dennison and Lovrein, 1997). Ammonium sulfate, however, needs to be removed by dialysis before electrophoresis may be used.Therefore, this not is not a convenient method for small volumes.
A slightly modified method, SDSIKCl precipitation was used for most proteins which were needed to be concentrated for electrophoresis. This method is unsuitable for general protein concentration as proteins would be denatured by SDS.
2.3.1.1 Reagents
5% Cm/v) SDS. SDS (0.5 g) was dissolved in 10 ml of ddH20.
3M KCl. KCl (2.24 g) was dissolved in 10 ml of ddH20.
2.3.1.2 Procedure
SDS [5% (v/v), 10 ).11] was addedto the sample(100 ).11) in a 1.5 ml Eppendorf tube.The solution was mixedby inverting the tube and 3 M KCl (10 ).11) was added. The mixture was again inverted and centrifuged (12 000 x g, 2 min, RT) and the supematant was discarded. For SDS-PAGE the precipitate may be dissolved in stacking gel buffer (10 ).11) and reducing treatment buffer (10 ).11) (Section2.6.1.1).
2.3.2 Concentration by dialysis
Dialysis uses the principle of osmosis to desaltor effect a buffer change or may be used to concentrate a sample. Dissolved molecules and dialysing solutions are separated by semi-permeablemembrane and movement of waterand ions is effected from the region of low ion concentration to the higher ion concentration. The selective sieving of the membrane due to its defined pore size also allows the movement of protein molecules of a certain size. The size at whichmolecules are retained by the membrane is called molecular cut off. When dialysis is used for desalting, distilled water or buffer with low ionic concentration may be used as the dialysis solution.This may cause influx of water and efflux of salts from the protein causing the membrane to swell. If the ionic strength of the buffer used is low or ddH20 is used to dialyse a solution a phenomenon called Donnan membrane effect may occur. The large size of proteins and the fact that they cannot pass through the pores causes an overall build-up of charge associated with the overall charge on the protein. To compensate this there is an influx of opposite charged ions. This may result in extremes of pH, eithercaused by influx of H+protons (acid pH) or OH- ions (alkaline pH). For this reason proteins should be dialysed against a buffer where possible as buffer ion dissociation and movement would compensate for ion inequalities across the membranes, correcting the pH to the desired value.
Maximal diffusion of solute and hence exchange of ions and H20 can be achieved only by frequently changing the dialysis buffer or H20 or dialyzing against large volumes.
The number of changes is less important than the total volume of dialysis buffer as large volumes maintain maximal concentration differences across the dialyzing membrane for longer (Englard and Seifter, 1990).
When used for concentration of highly diluted samples, the dialysis bag may be surrounded by solution of high concentration or with compounds with high affinity for H20 such as granular sucrose or PEG 20 000. Under these conditions water flows out from the membrane and dissolves in the granular or high concentration of the solute.
Sucrose may enter the bag contaminating the concentrated protein sample, whereas PEG 20 000 would not be able to do so due to its size.