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SDS-PAGE was carried out according to the method described by Walker (1996) with modifications as described below.

3.9.1Preparation of reagents

(a) Acrylamide stock solution

Acrylamide 30.0 g

Bis-acrylamide 0.8 g

Distilled water upto 100 ml

(b) Resolving gel buffer

1.5M Tris-HCl (pH 8.8)

(c) Stacking gel buffer

0.5M Tris-HCl (pH 6.8)

(d) Ammonium persulphate

10% solution (freshly prepared just before gel preparation) (e) N,N,N’,N’-tetramethylethylenediamine (TEMED)

(f) Sample buffer (2X)

0.5M Tris-HC1, pH 6.8 2.5 ml

10% SDS (w/v) 4.0 ml

Glycerol 2.0 ml

Bromophenol blue 2.0 mg

The Milli Q water was added to the buffer to a final volume of 10 ml. (g) Running Buffer (Tank buffer) 1X

Tris 3.0 g Glycine 14.4 g SDS 1.0 g

Milli Q water was added to a total volume of 1L. The pH of the buffer was 8.3.

(h) Staining solution

Coomassie brilliant blue R250 0.1 g Methanol 50.0 ml Acetic acid 10.0 ml Milli Q water was added to a final volume of 100ml.

(i) Destaining solution

3.9.2Preparation of resolving gel

Before preparation of the resolving gel, the spacer plate and the short glass plate were assembled together on the gel casting stand (Bio-Rad Laboratories, Hercules, CA, USA) with a spacing of 0.75 mm. The resolving gel was then prepared as follows with a final acrylamide concentration of 13% in the gel.

Resolving gel buffer 1.25 ml Acrylamide stock solution 2.15 ml Milli Q water 1.60 ml 10% APS solution 25.00 μl

TEMED 2.50 μl

The above reagents were mixed together in a flask by swirling, with APS and TEMED added just before casting the gel. The mix was then carefully poured between the glass plates of the gel cassette with care being taken to avoid incorporation of any air bubbles in the gel. A small volume of water was then distributed over the surface of gel, and the gel was allowed to polymerize (approximately 30 min).

3.9.3Preparation of stacking gel

The stacking gel was prepared as follows, with a final acrylamide concentration of 4% in the gel.

Stacking gel buffer 1.25 ml

Acrylamide stock solution 0.65 ml

Milli Q water 3.05 ml

10% APS solution 25.00 μl

TEMED 5.00 μl

The above reagents were mixed together in a flask by swirling, and approximately 20 μl was used to wash the surface of polymerized resolving gel (after decantation of surface water). The stacking gel mixture was then carefully poured over the resolving gel trying to avoid any air bubbles. A 10-tooth comb was then inserted and the gel was allowed to polymerize for about an hour.

3.9.4Preparation of the sample

with sample buffer in a 1:1 ratio. The mix was thoroughly vortexed and no heating of the sample was done.

3.9.5Preparation of molecular weight standards

Low range molecular weight standards (Bio-Rad Laboratories, Hercules, CA, USA) were used for estimation of molecular weight of the bacteriocin. The standards were prepared by diluting them in reducing sample buffer (prepared from stock sample buffer) in the ratio of 1:20. The composition of the stock sample buffer is as follows:

0.5M Tris-HCl (pH 6.8) 1.2 ml

Glycerol 1.0 ml

10% (w/v) SDS 2.0 ml 0.1% (w/v) Bromophenol blue 0.5 ml Distilled water 4.8 ml

The stock sample buffer was divided into 475 μl aliquots. For preparation of reducing sample buffer 25 μl of β-merceptethanol was added to an aliquot of sample buffer just before use. The standard was mixed with this reducing sample buffer in 1:20 ratio, and the mixture was heated at 95 °C for five minutes and then cooled to room temperature.

3.9.6Running the samples on the gel

After the polymerization of resolving and stacking gels, the comb was removed from the stacking gel and the gel cassette was assembled on the electrode assembly of the Bio-Rad Mini-PROTEAN® 3 electrophoresis cell (Bio-Rad Laboratories, Hercules, CA, USA). The assembly was then lowered into the Mini Tank of the cell. The inner chamber of the tank was filled with the running buffer. The outer chamber of the tank was then also filled with same running buffer.

Samples were then loaded in the wells (10-15 μl), with the molecular weight standards being loaded in one of the wells. The gel was then run at 100 V constant voltage for about 1.5 – 2.0 hours until the dye front reached the bottom of the gel. The gel cassette was then removed from the assembly and stained as described below.

3.9.7Staining and destaining of gel

After completion of the run the gel was carefully removed from the cassette, and soaked in coomassie blue staining solution for about 2 hours with gentle shaking. The staining solution was then replaced with destaining solution which was periodically replaced with fresh solution. Destaining was continued until the protein bands could easily be distinguished from the blue background.

3.9.8Preparation of zymogram SDS-PAGE gel

To detect the lytic activity of the bacteriocin, a parallel zymogram SDS-PAGE gel was prepared and run in a similar manner as described above. The only difference was that 0.2% (w/v) autoclaved cells of sensitive strain L. lactis ssp. cremoris 2144 were incorporated in the resolving gel as described by Potvin et al. (1988). Furthermore, after completion of the electrophoresis run, the gel was not stained with Coomassie blue but was renatured according to the method described by Beukes et al. (2000). Briefly, the gel was initially soaked in distilled water followed by treatment with renaturation buffer (25 mM Tris-HCl [pH 7.5] containing 0.1% Triton X-100 and 10 mM MgCl2) for 16 hours. Clear lytic bands were observed around the bacteriocin band against the opaque background of dead cells. The gel was compared with the Coomassie stained gel to locate the active band and determine its molecular mass.