Samples from flower of the Ixora javanica and fruit of the Mimosa Diplotricha have been grinded into powder form by Dr. Zazali. 30g of the ground plant material was dissolved in 250ml dichloromethane and shake overnight at 150rpm in room temperature at 25oC by using shaking incubator (SI-900R). Then, the solution was filtered by using Whatman filter paper and the residues are collected and dried under fume cupboard (FUMETEC). The residues are transferred into the conical flask and 250ml of 50% ethanol is added and shake overnight at 150rpm, 25 oC. Then, the mixture was filtered. Filtrate was collected and poured into a separation funnel. Dichloromethane is added into the separation funnel and shake the mixture of dichloromethane and ethanolic solution. The bottom layer of the dichloromethane is extracted out as waste. This is repeated for several times to remove the lipid of the plant material. Then, the ethanolic phase is collected. The ethanolic solution is then undergone evaporation by using rotary evaporator to evaporate the ethanol. The concentrated plant extract is then undergo freeze-dry and keep at -20 oC.
3.2 Column chromatography
Polyacylamide column is packed by added amide 6 that dissolved in the 2% acetic acid to the column bit by bit. 50centimeter of the amide is packed. Then, the column is connected to the akta prime plus fraction collector to calibrate the column with 2% acetic acid by setting the flow rate as 0.1ml/min and run overnight. Then, 1g of Ixora javanica extract was dissolved in 2ml 2% acetic acid was injected into column through the akta prime plus fraction collector. The flow rate was set to 1.5ml/min. The pressure was ensured not to more to 0.4 atm. The fraction was collected when the absorbance was increased dramatically. The colour pigmented compound was not collected although it will raise the aborbance.
3.3 Preparation of agar plate
Agar plates were prepared by using Mueller-Hinton agar. 20g of Muller-Hinton agar was suspended in 1000ml of distilled water in conical flask. The mixture was stirred until it completely dissolved in distilled water by using glass rod. Then, the mixture was sterilized by autoclave machine (Tomy SS-325) at 121 oC for 15 minutes. The mixture is then cooled to 40 oC after sterilized and poured into petri dishes and solidified became agar. The agar plates were incubated overnight in the incubator at 30 oC to ensure no any contamination occurred. The agar plates is keep in refrigerator for prolonged usage.
3.4 Cultivation of bacteria
Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus) and gram-negative (Plesiomonas shigelloides, Escherichia coli, E.coli 0157:H7, Salmonella sp., Salmonella typhi) bacteria were given in glycerol stock solution. Each bacteria species was streaked on the agar plate by using a loop. Cultivation of bacteria has done in laminar flow to ensure the surrounding area always in sterile conditions. The wire of the loop was heated until red-hot and cooled it before picking up bacteria. The bacteria were streaked on the agar surface using dilution streaking method. The loop was cleaned by glycerol 20% and heated red-hot each time before used or for the different species of bacteria to be cultivated. This is to prevent growth of other bacteria and contamination. The agar plates were then incubated in the incubator at room temperature for 24 to 48 hours.
3.5 Screening for antimicrobial activity of extracted protein from plant
Single colony of the B.subtilis was taken from the agar plate and transfered and streaked homogenously on a new agar plate by using a loop. 40µl of protein extracts were pipette dropped onto the agar plate that have been make 4 holes for the input of protein extracts. The agar plates were incubated in the incubator at room temperature for 24 hours. The same procedure was repeated for other bacteria. Another set of agar plates were used for antibiotics such as amphicilin, kanamycin and streptomycin. The result was observed.
3.6 SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis)
SDS-Page is method performed based on the discontinuous buffering system by Laemmli (1970).
3.6.1 Discontinuous Polyacrylamide Gels
The stacking gel and resolving gel solutions were prepared as below:
Solution
Stacking gel (4%)
Resolving gel (17%)
30% Arylamide/Bis
1.33ml
5.61ml
0.5M Tris-HCl, pH 6.8
2.5ml
-
1.5M Tris-HCl, pH8.8
-
2.5ml
10% SDS
0.10ml
0.10ml
18.3 MΩ-cm H2O
6.10ml
1.74ml
TEMED
10µl
5µl
10% APS
50 µl
50 µl
Total volume
10.09ml
10.005ml
Overlay solution was prepared as below:
Solution
Volume
10%(w/v) SDS
100µl
Distilled water
900µl
The resolving gel solution was prepared by mixing all the reagent except 10% APS and TEMED in a flask. The solution was degassed for 3 minutes. 10% APS and TEMED were added after degassed and swirled gently to mix and were poured in between the glass plates by using pipette. The solution is poured gently to prevent it from mixing with air and avoid formation of bubbles. The resolving gel is overlaid with 0.1%SDS or distilled water. The gel was allowed to polymerize for 1 hour. After polymerization, the overlay solution was poured.
The stacking gel monomer solution was prepared by mixed all the reagents as shown in the table except 10% APS and TEMED. The solution was degassed for 5 minutes before the 10% APS and TEMED were added. The solution is swirled gently after 10% APS and TEMED is added and poured on top of the resolving gel until the top of the plates by using pipette. The comb as inserted slowly and gently. The stacking gel was allowed to polymerise for 30-40 minutes. The comb is removed gently when it is to be used.
3.6.2 Sample loading
Sample buffer was prepared as shown below:
Solution
Volume
0.5M Tris-HCl,pH 0.25ml
0.25ml
Glycerol
0.40ml
10% SDS
0.40ml
β-mercaptoethanol
0.10ml
0.5% bromophenol blue
0.10ml
Distilled water
0.75ml
Total
2.00ml
Running buffer was prepared as table shown below:
Solution
Volume
10X Tris/Glycine/SDS
100ml
Distilled Water
900ml
Total
1000ml
The assembly was filled with running buffer until the below the edge of the outer gel plate. Sample is added with sample buffer with ratio 1:4 and heated at 95oC for 5 minutes. Samples and molecular weight markers were loaded slowly into the wells and allows them to settle at the bottom of the well. Electrophoresis was performed at a constant voltage of 120 V for around 2 to 3 hours. The electrophoresis was stopped when the tracking dye front was around 1cm from the bottom of the gel. The gel was stained with Coomassie Brilliant Blue.
3.6.3 Colloidal Coomassie Staining
Staining solution
5% (w/v) Coomassie Brilliant Blue G-250 0.1g in 2ml or 1.0g in 20ml
Phosphoric acid, H3PO4 11.8ml
Ammonium sulphate, (NH4)2SO4 100g
The staining solution prepared by the 5% (w/v) Commassie Brilliant Blue separately. 100g ammonium sulphate dissolved in distilled about 500ml. Phosphoric acid is added. 5% (w/v) Coomassie Brilliant Blue is added little by little. Then, the mixture is top up to 1L.
Destaining solution
Methanol 40ml
The staining solution was topped up with distilled water to make a total volume of 200ml.
Protein can be visualized directly in gel by Coomassie staining. Before staining, methanol is added to staining solution with the ratio 1:4. The gel immersed into the staining solution and shake overnight. The gel is destained in destaining solution with gently agitation on the shaker. The destaining solution was replenished several times until the background of the gel become clear and fully destained. The result is observed.
3.6.4 Silver staining
Silver staining was used to visualize the bands on the gel which the band is not clearly shown by colloidal coomassie staining. The protocol was modified from Heuckeshoven and Dernick (1985). The solutions were prepared according to the measurements given below:
Fixing solution
[40% (v/v) ethanol, 10% (v/v) acetic acid, 150ml]
Ethanol 60ml
Glacial acetic acid 15ml
Top up with distilled water to 150ml
Sensitizing solution
[30% (v/v) ethanol, 0.5% (v/v) glutaraldehyde, 8mM sodium thiosulphate, 0.5M sodium acetate, 150ml]
Ethanol
45ml
Glutaraldehyde*
0.75ml
Sodium thiosulphate
Sodium acetate
Top up with distilled water to
0.30g
10.2g
150ml
Silver solution
[0.04% (v/v) formaldehyde, 150ml]
Silver nitrate
Formaldehyde
Top up with distilled water to
0.375g
60µl
150ml
Developing solution
[0.24M sodium carbonate, 0.04% (v/v) formaldehyde, 150ml]
Sodium carbonate
Formaldehyde
Top up woth distilled water to
3.75g
60µl
150ml
Stop solution
[ 40mM EDTA-NA2 . 2H2O, 150ml]
EDTA-NA2 . 2H2O
Top up with distilled water to
2.19g
150ml
Preserving solution
[30% (v/v) ethanol, 4.6% (v/v) glycerol, 150ml]
Ethanol
Glycerol
Top up with distilled water to
45ml
6.9ml
150ml
*Optional
From the coomassie staining, the gel was immersed in sensitizing solution for 30 minutes. The sensitizing solution is discarded. Then the gel was washed. The gel washed 3 consecutive times with distilled water. Each wash lasted for 15minutes. The gel then was incubated in a silver solution for 20 minutes. The gel was washed twice with distilled water for a length of 1 minute per washing. After that, the gel was incubated in a developing solution and the gel is immersed in stopping solution after the desired band intensity is obtained. Stopping solution added for 10 minutes then the gel is again washed with distilled water for 3 times with each last for 10 minutes. After washed, the gel was preserved in preserving solution. The silver staining procedure was carried out on an orbital shaker which was shaked about 50 rpm.
Summary of the sequenc used in staining by the silver method
Step
Solution
Duration (min)
1
2
3
4
5
6
7
8
Fixing
Sensitizing
Washing
Silver
Developing
Stopping
Washing
Preserving
30
30
15 (3 times)
20
10
10
10 (3 times)
Few months
