9. Gel Filtration Chromatography



Separation of circulating filarial antigen (CFA-2) from Plasma


A. Principle :


Gel filtration is a separation technique useful for the purification of thousands of enzymes, polysaccharides, nucleic acids, proteins and other biological macromolecules. Gel  filtration permits the preparative separation of molecules by their native molecular weight due to the differential penetration of the gel matrix by molecules of different size. Very large molecules which never enter the gel matrix, move through the chromatographic bed fastest. Smaller molecules which can enter the gel pores, move more slowly through the column, since they spend a portion of their time in the stationary phase (gel). Molecules are therefore, eluted in order of decreasing molecular size.


B.Materials :


a.Pre-swollen Ultrogel ACA 34.


b.0.05 M SPB, pH 7.2   (Appendix- I)


c.Chromatography set up.


d.Circulating filarial antigen (CFA).   (Appendix- II)


e.Gel filtration molecular weight markers.




C. Method :


1.Transfer  about 100  ml  volume  of  pre-swollen ultrogel  ACA  34 to a vacuum flask  containing 50 ml of 0.05 M SPB, pH 7.2.


2.Mix with a glass rod and deaerate the gel suspension with the help of vacuum pump until the air bubbles stop coming out.


3.Pack  the chromatography  column  (LKB, Sweden) with bed dimension 1.6x 50 cm at  40C using either a peristaltic pump at a flow rate of 25 ml/hr or at a constant ydrostatic pressure of 70-100 cm H2O.


4.Equilibrate the column with deaerated 0.05 M SPB, pH 7.2 for 24 hr with a flow rate of 15 ml/hr.


5.Apply 1 ml of CFA (20 mg/ml) without injecting air bubbles to the gel and elute with 0.05 M SPB at a flow rate of 8 ml/hr.


6.Collect 2 ml fractions and read their absorbance at 280 nm wave length and record with automatic one channel recorder.


7.Pool fractions in each protein peak separately and concentrate by ultra filtration.


8.Check antigenic activity in pooled fractions by CIEP using FSIgG.


9.The second protein fraction showing filarial antigens is designated as CFA2.

Determination of molecular weight

1.Equilibrate the column with 0.05 M SPB pH 7.2.


2.Apply a mixture of gel filtration molecular weight marker proteins.


3.Collect fractions and determine elution volumes (Ve) of each marker protein from the recorded peaks.


4.Determine void volume (Vo) by elution volume of Blue Dextran 2000.


5.Determine gel bed volume (Vt).


6.For each protein determine the value of  Kav.


7.Draw standard graph by plotting Kav against log molecular weight.


   Kav = Ve-Vo /  Vt-Vo




8.Calculate Kav for each protein peak of CFA Viz, CFA1, CFA2, CFA3 and CFA4.


9.From the standard graph estimate the approximate molecular weight for each peak.


References :

1.Reddy,  M.V.R., Prasad,   G. B. K. S.  and  Harinath, B. C. Isolation and evaluation of antigens  from  microfilaraemic plasma and immunecomplexes for Diagnosis of bancroftian filariasis. Indian J. Pathol, Microbiol. 29 (1986), 179.


2.Gel Filtration Theory and Practice. Pharmacia Fine Chemicals, 1981.