EXPERIMENTAL
ANIMALS:
Albino rats of Wistar strain (100-150mg)
purchased from The King Institute, Guindy, Chennai and Tamil Nadu Animal
Science University, Chennai; were
received at 5 weeks of age and housed for acclimatization, at the animal house,
Department of Biochemistry, Madras University; until the start of the experiment.
The animals were weighed periodically, food pellets and water provided
uniformly, and housed under hygienic conditions. At the end of the treatment period
all the experimental animals were sacrificed by painless procedures and the
tissues used for studying the biochemical and marker proteins difference in
stress.
STRESSING
PROCEDURE:
To induce stress, rats were
exposed to bright light. In the
present study we attempted to manipulate the aversiveness of the test
conditions by comparing undisturbed control and gently handled Wistar rats with
rats that were provided with small space and exposed to high-light (1000–1200
lx) illumination in the box. The light intensities used in this study were
comparable to previous behavioral studies.
EXPERIMENTAL
DESIGN:
Prior to the test day, all rats were weighed and handled daily for 2 min
on 5 consecutive days to familiarize them with the general procedure involved
and to increase the stability of behavioral responses. Each subject was randomly
assigned to one of the two groups: i.e. control (CO), high-light test (HA) (providing
small space and HL: 1000–1200 lx illumination in the box). CO rats were left
undisturbed on the test day. For the HA group, the cage was moved from the
holding room to the adjacent test room, carry over experiments and put back immediately and returned to the
holding room. Two groups were tested per day (for a total of 14 days) between
9:00 a.m. and 1:00 p.m. and between 7:00 p.m. and 9:00 p.m. During the stress
procedure animals were deprived of food and water.
FOOD INTAKE:
A weighed amount
of food was placed in the hooper of the cage. The amount of food consumed was
noted by subtracting the amount of food left. Food intake was calculated in
terms of gm/100 gm body weight.
GROWTH RATE:
The
animals were weighed daily before the stress experiment. Daily changes of
growth rate were calculated.
TISSUE
PREPARATION:
250 mg of the fore, mid and hind brain tissue was
weighed, uniformly homogenized in an ice cold aqueous solution of 0.20 M sucrose containing 1 mM EDTA, 1 mM β-mercaptoethanol
(β-SH), 0.25 mM phenylmethane sulphonyl fluoride (PMSF) (1 ml medium / 0.250 g tissue)., and the homogenate was
used for the isolation of myelin sheath. Myelin sheath was isolated by density gradient
centrifugation technique. 20 µl of myelin diluted to 0.1 ml was used for the
assay.
ISOLATION OF
MYELIN SHEATH:
Myelin sheath was isolated
from neuronal axons of fore, mid and
hind brain tissue by sucrose density gradient centrifugation by the method of Norton and
Poduslo, (1973).
Reagents:
Sol A (For Homogenization):-0.2M Sucrose, 1mM EDTA, 1mM β –SH and 0.25mM PMS.
Sol B :- 0.32M Sucrose and 0.78M Sucrose, each containing 1mM EDTA,1mM β –SH and 0.25mM PMSF.
Procedure:
Fresh
brain tissue is suspended in an ice cold 0.20M sucrose containing 1 mM EDTA, 1 mM β-SH, 0.25 mM PMS.
The suspended tissue was gently disrupted by up and down strokes in a Teflon glass
homogenizer. A 1.0ml aliquot
of this
suspension was layered on 0.5 ml of 0.32 M
sucrose and 0.1 ml of 0.78 M sucrose respectively (each containing 1 mM
EDTA, 1 mM β-SH and 0.25 mM PMSF) and centrifuged in an ultracentrifuge at 106000 g for
30 min at 4°C, with slow acceleration and no brake. The crude myelin fraction
at the interface between 0.32 and 0.78 M sucrose was recovered and washed twice
with deionized water.
The
myelin isolated was weighed and expressed in mg / 250mg of tissue.
BIOCHEMICAL ASSAYS:
ESTIMATION OF
PROTEIN:
The
total protein content was estimated by the method of Lowry et al., (1951).
Reagents:
Solution
A
2%
Sodium Carbonate in 0.1N Sodium Hydroxide
Solution
B
0.5% Copper Sulphate in 1% Sodium
Potassium Tartarate.
Alkaline Copper Reagent
50 ml Solution A + 0.5 ml Solution B
Folin’s Phenol Reagent
Commercially available Folin’s Phenol was diluted with water in the ratio 1:2 before
use.
Standard
10
mg Bovine Serum Albumin in 100 ml water.
Procedure:
0.1 ml of the sample was
made up to 1.0 ml with water, added 4.5 ml of alkaline copper
reagent, shaken well and allowed to stand for 20 minutes. Then, 0.5 ml of Folin’s
Phenol reagent was added, mixed well and incubated at 370C, for 15
minutes. Standards containing bovine serum albumin at concentrations from 20 µg to 100 µg were treated similarly. The
blue colour developed was read at 620 nm in a Shimadzu UV Visible double beam
spectrophotometer.
The total protein content was expressed in % / mg
of myelin
ESTIMATION OF
ATPase ENZYMES:
Na2+ -
K2+ ATPase:
The
total sodium-potassium - ATPase activity was estimated by the method 0f
Bonting, (1970).
Reagents:
Tris Hcl Buffer
184mM; 1.113 g of tris Hcl in 50.0
ml of distilled water, pH 7.5Magnesium Sulphate :-50.0
mM; 369.9 mg magnesium sulphate in 25.0 ml of distilled water.
50.0 mM; 878.0 mg sodium chloride in 25.0 ml distilled water.
Potassium Chloride
50.0 mM; 372.5 mg potassium chloride in100.0ml
distilled water
Ethylene diamine tetra
1.0 mM, 9.3 mg of ethylene diamine tetra acetic acid
in 25.0 ml of distilled water.
Adenosine Triphosphate
22.0 mg of adenosine triphosphate in 1.0ml of distilled water.
ANSA
150.0
g sodium bisulphate + 5.0 g sodium sulphite + 800 ml distilled water, heated to
50oC ,added 2.5 g
1-amino-2-naphthol-4- sulphonic acid, dissolved well, made up to 1250.0
ml with distilled water, filtered and
stored.
Ammonium Molybdate
30.0 mg ammonium molybdate in 1.0 ml distilled water.
Procedure:
To 0.1 ml of the sample, added 1.0 ml of Tris-Hcl buffer,
0.2 ml of magnesium sulphate, 0.2 ml of potassium chloride, 0.2 ml of sodium
chloride, 0.2 ml of ethylene diamine tetra acetic acid, 0.2 ml of adenosine
triphosphate, shaken well and incubated for 10 minutes at 370C. 10%
trichloroacetic acid was then added, shaken well to stop the reaction, added
1.2 ml of distilled water, 0.5 ml ammonium molybdate, 0.2ml of ANSA, shaken
well and incubated at 37oC for 15 minutes. The optical density was
read at 640 nm in a Shimadzu UV Visible double beam spectrophotometer.
The total sodium-potassium-ATPase activity was expressed in
units/min/ mg of myelin.
Ca2+
ATPase: (E.C.3.6.1.38)
The
activity of Membrane Ca2+ ATPase was determined by the method of Hjerten
and Pan, (1983).
eagents:
1.
Tris - Hcl buffer : 125mM
2.
CaCl2 : 50mM
3.
ATP : 10mM
4.
TCA : 10%
Procedure:
The
reaction mixture containing 0.1ml of buffer, CaCl2, ATP and with
0.1ml of membrane preparation was mixed well and incubated at 37°C for 15mins.
The reaction was arrested by the addition of 1.0ml TCA and centrifuged. The
supernatant was taken for estimation of phosphorous by Fiske & Subbarow
method.
The
enzyme activity was expressed as µ moles of phosphorous liberated/ min/ mg of
myelin.
Mg2+
ATPase (E.C.3.6.3.1)
The
activity of Mg2+ ATPase was determined by the method of Ohiniso et al., (1982).
Reagents:
1.
Tris - Hcl buffer : 37mM
2.
MgCl2 : 25mM
3.
ATP : 10mM
4.
TCA : 10%
Procedure:
The
reaction mixture containing 0.1ml of buffer, MgCl2, ATP and with
0.1ml of membrane preparation was mixed well and incubated at 37°C for 15mins.
The reaction was arrested by the addition of 1.0ml TCA and centrifuged. The
supernatant was taken for estimation of phosphorous by Fiske & Subbarow
method.
The enzyme activity was expressed as µ
moles of phosphorous liberated/ min/mg of myelin.
ESTIMATION OF
PROTEIN BOUND CARBOHYDRATES:
Delipidised
residues of tissues were prepared according to the method of Folch et al., (1951).
Reagent:
1.
Chloroform-Methanol mixture 2:1(v/v)
2. Saline -0.89%
Procedure:
The
tissue were washed with saline and dried with a filter paper and weighed about
500mg and homogenized with 7ml of methanol in a potter elvehjem homogenizer and
filtered through a whatman No 1 filter paper into a conical flask. The residue
after filtration was scraped and homogenized in 14ml of chloroform. The residue
was scraped from the filter paper and preserved for glycoprotein estimation.
A
known amount of delipidised residues of the tissues were hydrolyzed with 2.0ml
of 4N HCl at 100˚C for 4 hours. The hydrolyzed materials was neutralized with
KOH and used for the estimation of hexose, hexoseamine and fucose.
ESTIMATION OF
HEXOSE:
Hexose was estimated by
the method of Niebes (1972).
Reagents:
1. Orcinol-sulphuric acid reagent:
Reagent - A: sulphuric acid-water mixture (3:2v/v)
Reagent - B: 1.6g of orcinol in 100mlof water
Reagent A and B was mixed in the
ratio of (15:2v/v)
2. Standard:
50g of galactose and 50mg of mannose were
dissolved in 100ml of distilled water. This solution was diluted to 1:10
proportion to give a concentration of hexose 100µg/ml.
Procedure:
To
0.05ml of neutralized samples, 0.5ml of distilled water was added. Then 8.5ml
of orcinol reagent was added to all the tubes placed in a cold water bath very
slowly. The contents were mixed well and the tubes were heated at 80˚C for 15
min. Then the colour developed in the dark, after cooling was read at 540nm
using a photochem colorimeter.
Standard
solutions containing 25-100µg of hexose were treated in a similar manner to
obtain a standard curve for comparison.
The
hexose content was expressed as mg/g of defatted tissues
ESTIMATION OF
HEXOSAMINE:
Hexosamine
was estimated by the method of Wagner (1979).
Reagents:
1. Acetyl acetone reagent.
Reagent A : 1N trisodium phosphate.
Reagent B : 0.5N potassium tetra borate.
2. Acetyl acetone 3.5% was prepared by mixing reagent A and B in
the ratio 98:2v/v.
3. Ehrlich’s reagent: 320mg of
para dimethyl amino benzaldehyde was dissolved in 21ml of isopropanol and 3.0ml
of concentrated hydrochloric acid.
Procedure:
0.5ml of the neutralized sample was made up to 1.0ml.
Standard galactosamine (in the range of 10 to 40µg) was also made up to 10ml.
Blank comprised of 1.0ml of water 0.6ml of acetyl acetone reagent was added to
all the tubes and heated in a boiling water bath for about 30min. After cooling
2.0mlof Ehrlich’s reagent was added and the contents were shaken well. The pink
colour developed was read at 540nm against a water blank using a photochem
colorimeter.
Hexosamine
content was expressed as mg/g of defatted tissues.
ESTIMATION OF
FUCOSE:
Fucose content was estimated by the method of
Winzler (1955).
Reagents:
1.
sulphuric acid reagent : water (1:1)
(v/v)
2.
cysteine – HCl : 3%
3.
NaOH :
0.1N
4.
Standard fucose : 100mg
fucose in 100ml of distilled water.
Procedure:
To
50µl of hydrolyzed sample 2ml of alcohol was added, homogenized and
centrifuged. To the precipitate added 1ml of 0.1N NaOH and 4.5ml of sulphuric
acid and water (1:1) was added and the tubes were kept in ice bath. Then the
tubes were heated in water bath for 30 minutes. 0.1ml of 3% Cysteine-HCl was
added and mixed well. The tubes were left in the dark at room temperature for
75 minutes. Similarly blank and standard were carried out. The colour developed
was read at 420nm.
Fucose
levels are expressed as mg/g defatted tissue.
ESTIMATION OF SIALIC
ACID:
The estimation of Sialic acid was
determined by the method of warren (1959).
Reagents:
1.
Sulphuric acid : 0.1N.
2.
Periodate : 0.25M
in 0.1N sulphuric acid.
3.
Sodium arsenite : 4% in
0.5N hydrochloric acid.
4.
Thiobarbituric acid:
0.1N;
144mg of thiobarbituric acid was dissolved in10ml of water. The pH of the solution
was adjusted to
9.0with 6N sulphuric acid. The reagent was prepared
a fresh.
5.
Acidified butanol : 95ml of
n-butanol was mixed with 5.0ml of hydrochloric
acid.
6.
Standard Sialic acid:
10mg of
n-acetyl neuraminic acid was dissolved in 100ml of distilled water.
Procedure:
To 0.5ml of neutralized sample 0.24ml of periodates was added
and incubated at 37˚C for 30min. After incubation the reaction was arrested by
the addition of 0.25ml of arsenate. The tubes were shaken well and 2.0ml of
thiobarbituric acid reagent was added and the tubes heated in a boiling water
bath for 6min. After cooling 5.0ml of acidified butanol was added and the
butanol phase was separated after shaking well. The absorption was read at
540nm against a blank treated in a similar way using a photochem colorimeter.
Standard solutions containing 10-50µg n-acetyl neuraminic acid were also
treated similarly.
Sialic acid content was expressed as mg/g
defatted tissues.
SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE):
Whole brain homogenates were analysed by
SDS-PAGE to ascertain the relative protein concentration. Proteins were
analysed according to the method of Laemmli (1970).
Principle:
SDS-PAGE is the most widely used technique
to resolve the proteins according to their molecular weights using, acrylamide
cross linking agent N, N’- Methylene Bisacrylamide, in the presence of a free radical generator
and a catalyst Ammonium per sulphate and TEMED. The get polymerized into a
uniform 3-D net work with pores. The relative proportion of acrylamide and
bisacrylamide determines the porosity of the gel. In this method the protein,
irrespective of their initial charges are conferred by uniform negative by SDS.
SDS is an anionic detergent that strongly binds to denature proteins. The multi
subunit proteins are separated into their individual polypeptide components and
the polypeptide are rendered linear by the concerted effect of heat, SDS and
β-mercaptoethanol. The protein, which acquires negative charge and linear
nature as mentioned above, are electrophoresed under basic pH, towards the
anode. After electrophoresis the gel was stained with commassie brilliant blue.
Reagents:
1) 4X Running gel buffer (1.5M Tris pH 8.8):
45.375 gm of Tris was dissolved in 200 ml of
distilled water and pH was adjusted with 1N HCl, the volume was made up to 250
ml and stored.
2)
4X Stacking gel buffer (0.5M Tris pH 6.8):
15
gm of Tris was dissolved in water and pH was adjusted with 1N HCl before
adjusting the volume up to 250 ml.
3) 10% SDS in distilled water.
4)
Acrylamide-Bisacrylmide solution:
29gms
of Acrylamide (29%) and 1gm of bisacrylamide (1%) were dissolved in 100 ml of
distilled water. The solution was stirred well and stored at 4°C in a brown
bottle.
5)
10% APS in distilled water
(Freshly prepare everytime and kept at 4°C)
6)10X
Electrophoresis buffer (250Mm tris, 1.92M glycine and 1% SDS)
pH
8.3:
30.25gm of Tris,144gm glycine and 10gm of
SDS were dissolved in 800 ml of distilled water ,the volume was made up to 1000
ml and stored at room temperature.
7)1X
Electrophoresis buffer:
50
ml of 10X SDS Electrophoresis buffer was mixed with 450 ml of water.
8)10X
Transfer buffer (250mM Tris & 1.92 M glycine):
30.25gms
Tris and 144gms glycine were dissolved in 800 ml of distilled water .The volume
was made up to 1000ml and stored at room temperature.
9)1X
Transfer buffer with 20% methanol:
50ml of 10X buffer was mixed with 350 ml of
distilled water and 100 ml methanol and kept cold until used.
10)
2X Sample buffer:
672mg
EDTA was dissolved in a mixture of 12.5 ml stacking gel buffer,2.5 ml distilled
water,5ml bromophenol blue (10mg/ml in water ) and 10 ml of glycerol. The
solution was stirred well until the EDTA dissolved, 20 ml of 10% SDS was added
and the buffer stored at room
11)
Staining solution:
About
0.1% CBB and 10% methanol and 7% acetic acid was dissolved and made up to 100ml
using distilled water.
12)
Fixing solution:
10%
methanol and 7% acetic acid was dissolved and made up to 100 ml using distilled
water.
13)
Destaining solution:
25% methanol and 10% acetic acid was dissolved and made up to 100 using
distilled water.
PREPARATION OF GEL:
Running Gel or Seperating
Gel (12%):
Acrylamide
(30%) - 3.96 ml
Running gel
buffer - 3.0 ml
DD
H2O - 4.8
ml
SDS (10%) - 0.12
ml
APS (10%) - 0.12
ml
TEMED - 0.012 ml
Stacking gel (5%):
Acrylamide
(30%) - 0.5 ml
Stacking
gel buffer - 0.38 ml
DD H2O - 2.1 ml
SDS (10%) - 0.03 ml
APS (10%) - 0.03 ml
TEMED - 0.003 ml
Procedure:
Gel plates of dimension 17x17 cm with 1mm spacer were
set up to cast the gel. The resolving gel solution was poured in between the
two glasses and allowed to polymerise for 20 min. After resolving gel has
polymerized, 5% stacking gel solution were poured overlaying the resolving gel.
The comb was fixed properly to get clear out wells and the stacking gel allowed
polymerizing for 20 min. after confirming that the stacking gel had set
properly, the comb was removed slowly and the set up was placed in electrophoretic
apparatus.
Each lane of polymerized gel was loaded
with 50 µg of protein of experimental sample. The electophoresis was conducted
at a constant voltage of 50V and 10A current was supplied by means of a power
pack. The anode and cathode buffer tank was filled with reservoir buffer and
checked regularly for the migration of BPB dye towards anode. The current and
the respective voltage may be switched to 20 A and 100V after the sample
entered the resolving gel and the run was stopped when the tracking dye reached
the bottom of the gel. Then the gel was taken out of the plate and fixed using
fixing solution, and stained using staining solution for 4 hours. Then the gel
was subjected to destain using destaining solution to visualize the clear band.
PAS STAINING:
Fixing and
staining solutions:
1.
Periodic acid:
1g
of periodic acid was dissolved in 100ml of 3%acetic acid. Prepare freshly.
2. Schiff’s reagent:
1g of basic fusion was dissolved in 200ml of boiling
distilled water, mixed well and cooled to 50c. The solution was filtered and
and to the filtrate 20ml of 1N HCL was added. The contents were cooled at 25c
and 1.0g of potassium metabisulphite was added. The solutions was allowed to
stand in the dark, for 1 hour. 2.0g of activated charcoal was added and the
mixture shaken vigorously. After filtration the colourless solution was stored
at 0-4c. The solution was brought to room temperature prior.
Procedure:
Steps
in the treatment of the gel for glycoprotein staining are:
Step Gel treatment at 25 C Time
of minutes
1. Immersed in 12.5 % TCA` 30
2. Rinse with distilled water 0.5
3. Immersed in periodic acid solution 50
4. Wash with several
changes of distilled water 0.2
5. Immersed in the staining solution for
dark 50
6. Wash thrice with
freshly prepared 0.5%
Potassium bisulphate 30
7. Wash with
distilled water until excess stain was removed 0.2
Finally the gel was stored in 5% acetic acid
solution.
STATISTICAL
ANALYSIS:
The data obtained in
the experiments were analysed by one-way ANOVA analysis by utilizing SPSS
package. The results of the experiments were expressed as mean SD. The level of
significance was set at p< 0.05.
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