14. Fast Protein Liquid Chromatography (FPLC)


A. Principle:


            FPLC system is a complete system for laboratory scale chromatographic separations   of proteins and other biomolecules. This system consists of a programmable controller for developing and controlling automatic separation procedures, one or more pumps for liquid delivery, a mixer to ensure accurate and reproducible elution gradients, valves for sample injection and flow path control, one or more monitors for measuring chromatographic profile, a recorder for documenting chromatographic profile, a fraction collector and a chromatography rack for mounting the component in a compact laboratory bench top arrangement.


In this experiment a partially purified Mycobacterium tuberculosis H37Ra strain    excretory-secretory protein i.e, EST-G2 will be further purified using a cation exchange, Resource 'S' 1 ml column (Amersham Pharmacia Biotech, Sweden). The column is first equilibrated using a 5:2 ratio of buffer A (sodium citrate 0.1 M, pH 3.6) and buffer B (sodium citrate 0.1 M + 2 M sodium chloride, pH 3.6) at a flow rate of 1 ml  per minute. The loop is filled with sample (50 µg/ 200 µl). The separation was achieved in run mode using a preprogrammed automatic separation procedure. As per the  programme after injection of the sample, it was eluted at a flow rate of 1 ml per minute with the proportion of B was increasing linearly to the ratio of buffer B:A of 20:80 at  2 minute and of 100:0 at 7.6 min. fractions of 0.5 ml volume collected were checked  for tubercular antigenic activity by indirect ELISA.