MATERIAL AND METHOD
3.1 Protease from biological (Bacteria and Plant) sources-Sample collection
3.1.1 Soil Collection
Soil was collected in sterilized sampling bags of Ghazipur poultry waste site, India. Soil was
selected from areas where naturally degraded feather were seen. The samples were brought to
the laboratory and processed for the analysis the same day. Soil samples (10%) were
suspended in basal media (PeptoneBroth - Beef extract 3g/l Peptone 5g/l, NaCl 75g/l,
Distilled water 1liter) and kept for growth at 37°C for 6 days. After 24 hrs of incubation at
regular intervals of 6 hrs the activity of protease was measured and sample showing
maximum activity was screened for protease producing strains.
3.1.2 Plant Material Collection
Green and Senesced leaves of different plants growing in and around the University area in
Noida, Uttar Pradesh, India were collected in forenoon in plastic bags and brought to the
laboratory and processed for the analysis the same day.
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3.2 Isolation and Screening of protease-producing strains and plants
3.2.1 Soil Isolate
Soil isolate showing maximum protease activity were plated on skim milk agar (skim milk
powder 10 %, peptone 0.1%, NaCl 0.5% and 2 % agar). Plates were incubated at 37°C for 24
hours and the colony that showed clear zone were selected and was sub cultured in LB broth
The bacterial isolate was further incubated in cultivation media for 48 hrs and checked for
protease production.
3.2.2 Plant Source
Green and Senesced leaves of Asparagus officinalis (Asparagus), Ocimum sanctum (Tulsi),
Murraya koenigii (Karipatta), Tagetes erecta (Marigold), Hibiscus rosasinensis (China rose),
Carica papaya (Papaya), Vinca Rosea (Periwinkle), Azadirachta indica (Neem), Musa
paradisiaca (Banana), Lantana camara (Lantana) growing in and around the University area
in Noida Uttar Pradesh India were collected in forenoon in plastic bags and brought to the
laboratory and processed for the analysis the same day. Leaves of each plant were washed
with distilled water and blotted dry were weighed (15 gm), homogenized with mortar and
pestle in three volumes w/v 10 mM Tris-chloride buffer (pH 8.0) followed by filteration with
three layered cheese cloth. Then the filterate was centrifuged at 10,000 rpm for 10 min at
4°C. The supernatant of leaves of each plant was collected separately and used as crude
preparation. The crude extract was checked for protease activity.
3.3 Identification of soil isolates and plant species
3.3.1 The isolate GW1 was identified originally as a strain of pseudomonas based on
Morphological, Biochemical and 16S rDNA sequencing characteristics.
3.3.2 Lantana camara was selected as a biological source for protease isolation and it is
classified under family verbanaceae genus Lantana species camara according to Linnaeus
classification system.
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3.4 Identification and classification of soil isolate
3.4.1 Microscopic Examination
3.4.1.1 Gram Staining
A drop of sterile saline water was placed on the microscope slide. A light suspension of the
test culture was made with normal saline and heat fixed. The slide was allowed to cool and
flooded with crystal violet for 60 seconds and then washed with distilled water. It was then
flooded with Gram’s iodine for 60 seconds, washed with water and excess water was drained
off. The stain was then decolorized with acetone which was washed off immediately.
Counterstaining was done with safranin for 2 minutes. The slide was then washed, air dried
and observed under a microscope.
3.4.1.2 Endospore Staining
A smear of the culture was prepared on a clean slide and heat fixed. The smear was flooded
and kept saturated with 5% malachite green stain while the slide was heated continuously for
5 minutes. The smear was then rinsed with distilled water and counter stained with safranin
for 2 minutes. The slide was once again washed with water, air dried and investigated under a
microscope for endospore formation.
3.4.2 Biochemical Test
3.4.2.1 Oxygen Requirement
The bacterium was inoculated into nutrient broth (peptone-5g/l, NaCl-3g/l, Beef extract-3g/l,
distilled water-1liter) to study the oxygen requirement of the pure isolate. Depending on the
growth of the bacteria are grouped as aerobic, micro-aerobic and anaerobic.
3.4.2.2 IMViC Test
IMViC is an acronym that stands for indole, methyl red, Voges-Proskauer, and citrate test.
51
3.4.2.2.1 Indole Production
The bacterial isolate was inoculated into Tryptone broth (tryptone-10g/l, NaCl-5g/l, distilled
water-1 liter) and incubated at 37°C for 48 hours. Then 0.5 ml of Kovac’s reagent was added
and shook the culture gently. The test tube was observed for colour reaction.
3.4.2.2.2 Methyl Red Test
Bacteria produce acid by fermentation of glucose, which changes the pH of the medium. It
falls and maintained below 4.5. This test detects the production of acid. The isolated test
sample was inoculated in glucose phosphate broth (Protease peptone-5g/l, D-glucose-5g/l,
Na2HPO4-5g/l, distilled water-1liter) and incubated at 37°C for 2 days. Five drops of 0.04%
solution of alcoholic methyl red solution were added to the culture, mixed well and the results
were read immediately.
3.4.2.2.3 Voges-Proskauer Test
The isolated test sample was inoculated into 5ml glucose phosphate broth (Protease peptone-
5g/l, D-glucose-5g/l, Na2HPO4-5g/l, distilled water-1liter) and incubated at 37°C for 48
hours. Then 1ml of 40% potassium hydroxide (KOH) containing 0.3% creatine and 3ml of
5% solution of α-napthol in absolute alcohol were added. The results were read immediately.
3.4.2.2.4 Citrate Utilization Test
This test is used to study the ability of an organism to utilize citrate present in Simmon’s
media (MgSO4-0.2g/l, NH4H2PO4-1g/l, K2HPO4-1g/l, sodium citrate-2g/l, NaCl-5g/l,
Bromothymol blue-0.08ml, agar-15g/l, distilled water-1liter) as a sole source of carbon for
growth. The Simmon’s media was prepared, sterilized and poured into the tubes. The media
was allowed to cool and solidify in the form of slants. The slant was then stabbed with the
isolated bacterial sample using a needle and incubated at 37°C for 36 hours.
3.4.2.3 Nitrate Reduction Test
The enzyme nitrate reductase reduces nitrate to nitrite, ammonia, nitrous oxide, nitrogen, etc.
This test is used to detect the production of nitrate reductase. The isolated test organism was
inoculated in 5ml of nitrate broth (beef extract-3g/l, peptone-5g/l, NaCl-5g/l, potassium
nitrate-1g/l, distilled water-1 liter) and incubated at 37°C for 96 hours. Then we added 1ml of
52
α-naphthylamine reagent and 1ml of sulphanilamide reagent and the results were immediately
read.
3.4.2.4 Motility Test
This test used to check for the ability of bacteria to migrate away from a line of inoculation
due to physical features like flagella. The motility media for the motility test was prepared,
sterilized and poured into tubes. The media was allowed to cool and solidify in the form of
slants. The slants were then stabbed with the isolated bacterial sample using an inoculation
needle and incubated at 37°C for 24 hours.
3.4.2.5 Catalase test
One drop of 30% hydrogen peroxide was placed on a slide. One loopful of the fresh bacterial
culture was taken by a sterile needle and placed on the drop of hydrogen peroxide. Then
observed for bubble formation.
3.4.2.6 Oxidase Test
Fresh growth of bacterial isolate was scraped from the plate with the help of disposable stick
and rubbed on to the Impregnated oxidase test strip then the strip was observed for colour
reaction.
3.4.2.7 Amylase test
This test detects the ability of an organism to produce amylase enzyme for starch hydrolysis.
The bacterial isolate was inoculated on the starch agar plates (Beef Extract -3g/l, Peptone
-5g/l, NacL-5g/l, Agar-15g/l, 10% starch solution) and incubated at 37°C for 24 hours. After
incubation period the plates were then flooded with Gram’s iodine.
3.4.2.8 Urease Test
This test detects the ability of an organism to produce urease enzyme. The test organism was
inoculated on the slant of Urea agar and incubated at 37°C for 24 hours.
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3.4.2.9 Lactose Test
This test detects the ability of an organism to undergo lactose fermentation. The test
organism was inoculated into lactose broth (beef extract-3g/l, peptone-5g/l, lactose-5g/l) and
incubated at 37°C for 24 hours. The change in color was observed after adding phenol red
indicator.
3.4.2.10 Sucrose Test
This test detects the ability of an organism to undergo sucrose fermentation. The test
organism was inoculated into sucrose broth (beef extract-3g/l, peptone-5g/l, sucrose-5g/l) and
incubated at 37°C for 24 hours. The change in color was observed after adding phenol red
indicator.
3.4.2.11 Triple Sugar Iron Agar (TSI)
To determine the ability of an organism to attack a specific carbohydrate incorporated into a
basal growth medium, with or without the production of gas, along with the determination of
possible hydrogen sulphide (H2S) production.
TSI medium (Beef Extract – 3g/l, Yeast extract – 3g/l , Peptone - 15.0 g/l , Proteose peptone
-5.0 g/l, D-glucose monohydrate - 1.0 g/l , Lactose - 10.0 g/l, Sucrose - 10.0 g/l Ferrous
Sulphate (FeSO4.7H2O) - 0.2 g/l , Sodium Chloride - 5.0 g/l , Sodium thiosulphate
pentahydrate - 0.5 g/l, Phenol Red, 0.5 g/l aqueous solution- 48.0 ml , Agar - 12.0 g /l ) was
inoculated with an inoculating needle by stabbing the butt and streaking the slant. The tubes
were then incubated at 37°C for 24 hours.
3.4.2.12 Pseudomonas Agar P
Plates of Pseudomonas Agar P were prepared (Peptone-2%, potassium sulphate-1%, MgCl2 -
0.14%, Agar-1.8%) and the Bacterial isolate was streaked on Plates of Pseudomonas Agar P.
Then the plates were incubated at 37°C for 24 hours. After incubation period plates were
checked for pigment production for identification of isolate.
3.4.2.13 Pseudomonas Agar F
Plates of Pseudomonas Agar F were prepared (Peptone-1%, Proteose peptone- 1%, K2HPO4 –
0.15%, Agar- 1.5%, MgSO4). The bacterial isolate was streaked on Plates of Pseudomonas
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Agar F. Then the plates were incubated at 37°C for 24 hours. After incubation period plates
were checked for pigment production for identification of isolate.
3.4.2.14 Cetrimide Agar
Bacterial isolate was streaked on cetrimide agar plates (Peptone 20gm/l, MgCl2- 1.4gm/l,
potassium sulphate- 10gm/l, Cetrimethyl diammonium bromide- 0.3gm/l, Agar- 15gm/l,
Distilled water- 1 liter) and incubated at 37°C for 24 hours. After incubation period plates were
checked for pigment production for identification of isolate.
3.4.3 16S rDna Sequencing
The sample was sent to Bangalore GeneI, Bangalore, India for identification. Genomic DNA
was isolated from the pure culture pellet. Using consensus primers, the ~1.5 kb 16S rDNA
fragment was amplified using Taq DNA Polymerase. The PCR product was bi-directionally
sequenced using the forward, reverse and an internal primer. Sequence data was aligned and
analyzed for finding the closest homologs for the microbe.
3.5 Protease production
3.5.1 Protease Production from Bacteria
The cultivation media for protease production composed of (g L-1): Peptone, 5; Glucose, 10;
NaCl, 0.5; CaCl2.2H2O, 0.1; K2HPO4, 0.3; KH2PO4, 0.4; MgSO4 · 7H2O, 0.1; and yeast extract,
5. The pH was maintained at 7.5. Microbes were allowed to grow in 500 ml conical flask
containing 50 ml of the culture media that was maintained at 37°C at 140 rpm. 5% (v/v) of the
20 hrs old culture was inoculated in cultivation media. [213].
i. Tryptic soy broth (TSB)
ii. Gelatin (1%) + cultivation medium for protease production
iii. Casein (1%)+ cultivation medium for protease production
iv. Skim milk powder (1%)+ cultivation medium for protease production
v. Pigeon feathers (1%)+ cultivation medium for protease production
The above cultivation media were checked for enzyme activity by the modified method of
Tsuchida et al. [214].
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3.5.2 Protease Production from Plant
Senesced and green leaves
Green and Senesced leaves of Lantana camara (Lantana) growing in and around the
University area in Noida Uttar Pradesh India were collected in forenoon in plastic bags and
brought to the laboratory and processed for the analysis the same day. The leaves were weighed
after washing and rinsing with distilled water, homogenized with mortar and pestle in three
volumes wt/vol 10 mM Tris-chloride buffer (pH 8.0) followed by filteration with three layered
cheese cloth. Then the filtrate was centrifuged at 10,000 rpm for 10 min at 4°C. The
supernatant was collected and used as crude preparation. The crude extract was checked for
protease activity.
3.6 Protein EstimationProtein concentration was determined by the method of Bradford [215]. BSA (Bovine Serum
Albumin) was used as a standard protein for the estimation. A stock of BSA was prepared with
1mg / ml concentration Different dilutions of BSA was prepared (10µg/ml – 100µg/ml) and
added in each tube (total volume made upto 1 ml with distilled water). 5 ml of Bradford reagent
was added in each tube. After 10 min, OD was measured at 595 nm.
The absorbance against protein concentration was plotted to get a standard calibration curve.
The absorbance of unknown sample was checked and the concentration of the unknown sample
was determined by using the standard curve.
3.7 Determination of Protease Activity (plant and microbial source)Protease activity was assayed by a modified method of Tsuchida et al. [214] by using casein as
substrate. 100 μl of enzyme solution was added to 900 μl of substrate solution (2 mg/ml (w/v)
casein in 10 mM Tris–HCl buffer, pH 8.0).The mixture was incubated at 45°C for 30 min.
Reaction was terminated by the addition of an equal volume of 10% (w/v) chilled
trichloroacetic acid then the reaction mixture was allowed to stand in ice for 15 min to
precipitate the insoluble proteins. The supernatant was separated by centrifugation at 10,000
rpm for 10 min at 4°C. The acid soluble product in the supernatant was neutralized with 5 ml of
0.5 M Na2CO3 solution. The colour developed after adding 0.5 ml of 3-fold-diluted Folin–
Ciocalteau reagent was measured at 660 nm. All assays were done in triplicate. One protease
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unit is defined as the amount of enzyme that releases 1 µg of tyrosine per ml per minute under
the above assay conditions. The specific activity is expressed in the units of enzyme activity
per milligram of protein.
3.8 Protease Purification
3.8.1 Crude Extract Preparation
3.8.1.1 Bacterial Source
The bacterial strain was grown for 48 hrs at 37°C in the selected cultivation media. The
culture medium was centrifuged at 10,000 rpm for 10 min at 4°C and the cell-free
supernatant was collected.
3.8.1.2 Plant Source
Senesced leaves of Lantana camara was washed with distilled water and blotted dry were
weighed, homogenized with mortar and pestle in three volumes w/v 10 mM Tris-chloride
buffer (pH 8.0) followed by filteration with three layered cheese cloth. Then the filterate was
centrifuged at 10,000 rpm for 10 min at 4°C. The supernatant was collected and used as crude
preparation.
3.8.2 Ammonium Sulphate Precipitation
3.8.2.1 Bacterial Source
The supernatant was subjected for Ammonium sulphate precipitation. Supernatant was
precipitated with 0-60% ammonium sulphate. Ammonium sulphate was poured slowly into
the supernatant over a period of ten minutes, allowing the salt to slowly dissolve. The
supernatant was continually stirred at 4°C for an additional 1 hour. The precipitate was
collected by centrifugation (10,000 rpm for 10 minutes at 4ºC), and dissolved in a small
volume (1/50) of 10 mM Tris-HCl buffer (pH 8.0), and dialyzed against 4 liters of same
buffer for 12 hrs at 4°C. This step was repeated twice. The protease activity and protein
concentration was measured and specific activity calculated.
57
3.8.2.2 Plant Source
The crude enzyme preparation was precipitated with 0-30% and 30-60% ammonium sulphate
at 4°C, chilled for 1 hour at 4°C then centrifuged for 10 minutes (4°C) at 10,000 rpm. The
pellet was suspended in minimum volume of 10 mM Tris-Cl pH 8 and dialyzed against 6
liters Tris–chloride buffer (10mM, pH 8.0) at 4°C.
3.8.3 Chromatographic Techniques
3.8.3.1 Ion-Exchange Chromatography (for bacterial sources)
The dialyzed ammonium sulphate preparation was applied on a DEAE-cellulose column (2 X
24 cm) pre-equilibrated with 10 mM Tris-HCl (pH 8.0). The unadsorbed protein fraction was
eluted with the same buffer (150 ml). The enzyme was eluted with 2mM and 4 mM NaCl in
the same buffer at a flow rate of 1ml/min. The protease activity and protein concentration of
fractions was measured and specific activity calculated. Active fractions that contained
(80%) of the enzyme activity were pooled, and subsequently used for characterization. All
steps were conducted at 4°C.
3.8.3.2 Gel filtration Chromatography (for plant sources)
The protein pellet obtained after saturation with 0-30% ammonium sulphate was dissolved in
10mM Tris-HCl buffer and loaded onto a column of Sephadex G-250 (2 × 24 cm)
equilibrated with Tris-HCl buffer, pH 8.0. The one ml fractions were collected and checked
for protein and protease activity. From the activity profile, it was observed that the protease
was eluted as a well-resolved single peak of caseinase activity (11-20 ml fraction of one ml
each) coinciding with a single protein peak(11-20 ml fraction of one ml each) All the
fractions with high protease activities were pooled, dialyzed, and concentrated by
lyophilization and used for further studies.
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3.9 Electophoretic Techniques
3.9.1 Polyacrylamide Gel Electrophoresis (Bacterial and Plant Sources)
SDS-PAGE was performed on a slab gel containing 10% (w/v) polyacrylamide by the
method of Laemmli [216].
Resolving Gel (10%) Stacking Gel (4%)
Distilled Water- 4.1 ml Distilled water- 3.075ml
1.5M Tris (pH: 8.8)-2.5ml 1.5M Tris (pH:6.8)-1.25ml
10%SDS-50µl 10%SDS-25µl
Acrylamide-3.3 ml Acrylamide-670µl
10%ammonium persulphate-50µl 10%ammonium persulphate-25µl
Temed-5µl Temed-5µl
All the above reagent of resolving gel was mixed and after polymerization of the resolving
gel all the component of the stacking gel was mixed and poured inside the glass plate, wells
were made with the help of comb. After that protease sample was loaded in each well and
electrophoresed at 100V for 1:30 hour after electrophoresis completed, gel was removed from
in between the glass plates placed in the staining trough containing the staining solution,
stained for overnight on rocker. The gel was destained in destaining solution.
3.9.2 Zymography
Casein zymography was performed in 10% polyacrylamide slab gels containing SDS and
casein (0.12% w/v) as co-polymerized substrate, as described by Choi etal, [217]. After
electrophoresis, the gel was incubated for 30 minutes at room temperature on a gel rocker in
50 mM Tris-Cl (pH 7.4), which contained 2.5% Triton X-100 to remove SDS. The gel was
then incubated in a zymogram reaction buffer (30 mM Tris-HCl, pH 7.4, 200 mM NaCl and
10 mM CaCl2) left at 37°C for 12 hrs on rocker shaker. The gel was stained with Coomassie
brilliant blue (0.5% w/v) for 30 min. The activity band was observed as a clear colourless
area depleted of casein in the gel against the blue background when destained in 10%
methanol and 5% acetic acid for a limited period of time.
59
3.10 Characterization of Proteases (bacterial and plant source)
3.10.1 Hydrolysis of Protein Substrates
Protease activity with various protein substrates including casein, egg albumin, and gelatin
(2mg/ml) was assayed by mixing 2µg of the enzyme and 200 μl of assay buffer containing
the protein substrates. After incubation at 45°C for 30 minutes, the reaction was stopped by
adding 200 μl of 10% of chilled trichloroacetic acid (TCA) (w/v) and allowed to stand in ice
for 15 minutes. The undigested proteins were removed by filtration or centrifugation at
10,000 rpm for 5 min. and activity of the enzyme was measured.
3.10.2 Effect of pH on Enzyme Activity
The protease activity of the purified enzyme was measured at different pH values (3.0-12).
The pH was adjusted using the following buffers (0.2M) of Acetate (pH 2.0-4.0), phosphate
(pH 5.0-7.0), Tris-HCl (pH 8.0), and glycine-NaOH (pH 9.0-12.0). Reaction mixtures were
incubated at 45°C for 30 minutes and the activity of the enzyme was measured.
3.10.3 Effect of Temperature on Enzyme Activity and Stability
In bacterial source the activity of the enzyme was determined by incubating the reaction
mixture at different temperatures ranging from 20, 30, 40, 50, 60, 70 and 80°C were studied.
The activity of the enzyme was measured as described previously.
In plant source the activity of the enzyme was determined by incubating the reaction mixture
at different temperatures ranging from 30°C, 40°C, 50°C, 60°C, 70°C at different pH ranges
(pH 3.6, pH 5.0, pH 10.0, pH 11.0) were studied . The activity of the enzyme was measured.
3.10.4 Effect of Various Metal Ions on Protease Activity
In bacterial source the effects of metal ions on enzyme activity (e.g., Ca2+, Mg2+, Fe2+, Mn2+,
Zn2+, Hg2+, and Cu2+ [5 mM]) were investigated by adding them to the reaction mixture and
pre-incubated for 30 min at 45°C pH 10.0. The activity of the enzyme was measured as
described previously.
In plant source the effects of metal ions (e.g., Ca2+, Mg2+, Co+2, Fe+3, Mn2+ , Zn2+, Hg2+, and
Cu2+ [10mM]) were investigated by adding them to the reaction mixture and preincubated for
30 minutes at 45°C pH 10.0. The activity of the enzyme was measured.
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3.10.5 Effect of Protease Inhibitors and Organic Solvents on Enzyme Activity
The effect of various protease inhibitors (10mM) such as serine inhibitors
phenylmethylsulphonyl fluoride [PMSF],3,4-dichloroisocoumarin(3,4-DCI) and 4-(2-
aminoethyl)benzene sulfonyl fluoride (AEBSF), cysteine inhibitors E-64 and iodoacetic acid ,
Aspartic protease Pepstatin and Metalloprotease 1,10-Phenanthroline were determined by
preincubation with the enzyme solution for 30minutes at 45°C. The relative protease activity
was measured
The organic solvents used were Methanol, Ethyl acetate, Benzene, Glycerol, Sucrose,
Toluene, Acetone, Hexane, DMSO, Isopropanol and Ethanol. In the stability test, 1.0 ml of
organic solvent (100% v/v) was added to 1 ml of the reaction mixture and pre incubated at
37°C for 30 min. The remaining proteolytic activity was measured as described previously.
Stability was expressed as the remaining proteolytic activity relative to the solvent-free
controls (0%, v/v).
3.11 Industrial Application
3.11.1 Compatibility with Detergents
The compatibility of protease with laundry detergents was studied in the presence of 10mM
CoCl2. Detergents used were Henko (Henkel Spic, India), Surf, Surf Excel, Tide, Rin
(Hindustan Lever Ltd, India) and Ariel (Procter and Gamble, India). The detergents were
diluted in distilled water (0.7% w/v) and incubated with protease for 30 min at 60°C, and the
specific activity was determined. This was then compared with the control samples without
detergents.
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3.12 General methodology
3.12.1 Preparation of Dialysis Tubing
Dialysis tubing was cut into convenient length and boiled for 10 min in 500 ml of 2% sodium
bicarbonate and 1mM EDTA (pH 8.0) solution after that the tubing was rinsed thoroughly in
distilled water and boiled for 10 min in 1 mM EDTA then the tubing was allowed to cool and
stored at 4°C in 1mM EDTA buffer.
3.12.2 Preparation of Electrophoresis Reagents
Resolving Gel (10%) Stacking Gel (4%)
Distilled Water- 4.1 ml Distilled water- 3.075ml ml
1.5M Tris (pH: 8.8)-2.5ml 1.5M Tris (pH:6.8)-1.25ml
10%SDS-50µl 10%SDS-25µl
Acrylamide-3.3 ml Acrylamide-670µl
10%ammonium persulphate-50µl 10%ammonium persulphate-25µl
Temed-5µl Temed-5µl
Acrylamide- Bisacrylamide solution Staining Solution
Acrylamide – 30.0 gm Coomassie brilliant blue – 1.25 gm
Bisacrylamide – 0.8gm Glacial acetic acid – 35 ml
Distilled water – 100 ml Methanol - 200 ml
Final vol. - 500 ml (Distilled Water)
Destaining Solution Reservoir Buffer
Methanol- 45 ml Tris - 3.0 gm
Acetic Acid – 10 ml SDS – 1.0 gm
Distill Water – 45 ml Glycine – 14.4 gm
Distilled water – 1 liter
62
pH – 8.3
Sample Buffer (2X) Sample Buffer (2X)
1M Tris-Hcl (pH-6.8) – 12.5 ml for Zymography
SDS – 4.0 gm 0.5 M Tris-HCl, pH 6.8
β – mercaptoethanol – 10.0 ml SDS - 10
Glycerol – 20.0 ml Glycerol - 20%
1% Bromophenol blue – 4.0 ml Bromophenol blue - 0.5%
Final volume made up upto 500 ml with distilled water
Staining Solution for Zymography Destaining Solution for
Coomassie brilliant blue - 0.5% Zymography
Methanol - 10%
Acetic acid - 5%
Zymogram Reaction Buffer
30 mM Tris-HCl, pH 7.4,
200 mM NaCl,
10 mM CaCl2,
3.12.3 Preparation of Bradford Reagent
100mg of Coomassie brilliant Blue G250 dissolved in 50 ml of 95% ethanol then 100ml of
85% of Phosphoric acid was added. Final volume to 1 liter was made up with distilled water.
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3.12.4 Source of Chemicals, Media and Reagents
S.nos Name of chemical Source1 Acrylamide Sigma2 acetic acid CDH,Qualigens3 Acetone CDH,Qualigens4 Agar Hi Media5 Ammonium persulphate Sigma6 Ammonium sulphate CDH,Qualigens
74-(2-aminoethyl)benzene sulfonyl
fluoride Sigma8 Beef extract Sigma9 Benzene Sigma10 Bisacrylamide Sigma11 Bromophenol blue Sigma12 BSA Sigma13 Casein Sigma14 Cetrimide agar Hi Media15 CoCl2 CDH,Qualigens16 Coomassie brilliant blue R-250 Sigma17 Coomassie brilliant blueG-250 Sigma18 DEAE-cellulose Pharmacia19 3,4-dichloroisocoumarin Sigma20 D-glucose monohydrate CDH,Qualigens21 DMSO, Sigma22 E-64 Sigma23 Ethanol CDH,Qualigens24 Ethyl acetate CDH,Qualigens25 Ferrous Sulphate pentahydrate Merck26 Folin–Ciocalteau reagent Merck27 Gelatin Merck28 Glacial acetic acid CDH,Qualigens29 Glycerol CDH,Qualigens30 Glycine CDH,Qualigens31 Hexane CDH,Qualigens32 Hydrogen peroxide Merck33 Iodoacetic acid Sigma34 Isopropanol Merck35 K2HPO4 CDH,Qualigens36 KH2PO4, CDH,Qualigens37 Lactose CDH,Qualigens
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38 LB broth Hi Media39 Methanol Merck40 MgCl2 CDH,Qualigens41 MgSO4 CDH,Qualigens42 MgSO4 · 7H2O, CDH,Qualigens43 Motility Agar Hi Media44 Na2CO3 Merck45 NaOH CDH,Qualigens46 Pepstatin Sigma47 Peptone Hi Media48 1,10-Phenanthroline Sigma49 Phenol red CDH,Qualigens50 Phenylmethylsulphonyl fluoride Sigma51 Phosphoric acid CDH,Qualigens52 Potassium sulphate CDH,Qualigens53 Protein molecular weight marker Sigma54 Proteose peptone Hi Media55 SDS Sigma56 Sephadex G-250 Pharmacia57 Simmon’s media Hi Media58 Skim milk powder Hi Media59 Sodium Chloride CDH,Qualigens60 Sodium thiosulphate CDH,Qualigens61 Starch CDH,Qualigens62 Sucrose CDH,Qualigens63 Temed Sigma64 Toluene Merck65 Trichloroacetic acid CDH,Qualigens66 Tris Sigma67 Triton X-100 Sigma68 Tryptic soy broth Hi Media69 Urea agar Hi Media70 Yeast extract Hi Media71 β – mercaptoethanol Sigma
65