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.