Ion Exchange Chromatography Lab Report

3.9.3 Ion Exchange chromatography

As maximum activity was observed in the 50-80% fraction obtained from sequential ammonium sulfate precipitation, this fraction was subjected to ion-exchange chromatography on a DEAE-Cellulose column equilibrated with 0.15 M PBS. The resin was treated and column was packed as per the manufacturer’s instructions.

• 5gm dry resin was suspended in 25 ml of distilled water and left at room temperature for 30-45 minutes to settle down the matrix. The settled volume of the resin was measured and this was termed as Column volume (CV) and used as a measure for washing.

• Further the filter resin was suspended in 2 CV of 0.15 M NaOH containing 0.5 M NaCl for 10 minutes, and washing was continued for 5 CV of 0.15 M

The active fractions were pooled and extensively dialyzed against distilled water.

• The active elutes with highest peaks were tested for antimicrobial activity and stored at -20°C until required for further use and considered to be purified protein.

(Experiment was out sourced from Biotech Company and help of Dr Rakesh is duly acknowledged.)

3.9.4 Estimation of protein content

The protein concentrations of crude, ammonium sulfate precipitate and purified extract were determined by the Folin-Lowry assay (Lowry et al., 1951) using Bovine serum albumin (2 mg/ml)) as a standard. The absorbance at 540 nm was used to estimate protein content in aqueous, saturated fractions and purified extract.

3.9.5 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE):

The electrophoresis was performed according to Laemmli, (1971) using 10% acrlyamide. The gels were cast by mixing the ingredients as detailed below.

Table3.3: Ingredients of gel casting. Separating gel Stacking gel

Gel concentration (1%) 10ml 5ml

H2O 4.0ml 3.4ml

30% acrylamide mix 3.3ml 0.83ml

1.5 M Tris (pH 8.8) buffer 2.5ml 0.0ml

1.0 M Tris (pH6.8) buffer 0.0ml 0.63ml

10% SDS solution 0.1ml

The molecular mass was determined by comparison with the refrence protein markers (6.5-200 kD, Biorad 161-0317) with 9 bands; aprotinin (6 kDa), lysozyme (14.4 kDa), trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), bovine serum albumin (66.2 kDa), phosphorylase b (97.4 kDa), b-galactosidase (116.2 kDa) and myosin (200 kDa). Protein bands were visualized by silver staining.

3.10 Effect of Enzymes, pH and temperature on purified protein extracts:

3.10.1 Effect of Enzyme:

Two protein enzymes proteinase K (Fermentas, SKU # AM2546) and trypsin (Sigma, USA Cat # T 7409) were used to check their effect on antimicrobial activity of purified protein/peptide(s) using method by Carol et al.