Materials and Methods Final

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CHAPTER-4 MATERIALS AND METHODS

4. MATERIALS AND METHODS:

4.1. Plant collection & Processin:

Whole plant ofP. latifoliawere collected from Thirupati forest, Chitoor (Dist),Andhra Pradesh

(state) ,India during the month of Febraur!"#$ and the botanical identit was confirmed b

botanist of the Institute%The whole plant were shade dried at room temperature and made into

coarsepowder&'%

4.!. E"tracti#e #al$es:

The etractie alues were recorded in defferent solents with a iew to stud the distribution of

arious constituents of Premna latifolia% Accuratel weighed $%" g of coarsel powdered air*

dried material was placed in a glass*stoppered conical flas+ and macerated with #"" m of the

solent for - hrs, sha+ing fre.uentl, and then allowed to stand for #/ hrs% The miture was

filtered rapidl ta+ing care not to lose an solent% Twent*fie ml of the filtrate was transferred

to a tared flat*bottomed dish and eaporated to drness on a water bath%

The residue was dried at #"$0C for - h, cooled in a desiccator for &" min, and weighed

without dela%

1tractie alue ( 2) 3W2W1

Weight of drugtaken 4#""

5ere,

W#3 1mpt porceline dish

W! 3 Dish with etract

DEPT.OF ANIMAL BIOLOGY, SLS , HCU, HYD. DEPT.OF PHARMACOGNOCY, CPS, IST, JNTU,

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Ta%le.4.1: E"tracti#e #al$es o 'ierent sol#ents

Sol#ent Maceration ti(e )1*#al$e + )!*#al$e+ , E"tracti#e #al$e

Pet %ether !' hrs% -6%!6 g -6%g "%/ 2

1thl acetate !' hrs% $/%-$g $/%7!g !%/2

Acetone !' hrs% $/%6#g $/%6$g #%-2

8ethanol !' hrs% -$%&&g -$%''g '%'2

Chloroform !' hrs% $/%-$g $/%-7g "%&'2

9*butanol !' hrs% $/%6#g $/%6'g "%$2

Ccloheane !' hrs% -6%!6g -6%&"g "%'2


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i.4.1:

E"tracts it/ 'ierent sol#ents

4.0. Met/o' o e"traction

1traction was done b sohlet method% In this method normall a solid material

containing some of the whole plant dried powder form material is placed inside a thimble made

from thic+ filter paper, which is loaded into the main chamber of the :ohlet etractor% The

etraction solent to be used is ta+en into a distillation flas+ and the :ohlet etractor is now

placed onto this flas+% The :ohlet is then e.uipped with a condenser%


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The solent is heated to reflu% The solent apour traels up a distillation arm, and

floods into the chamber housing the thimble of solid% The condenser ensures that an solent

apour cools, and drips bac+ down into the chamber housing the solid material%

The chamber containing the solid material is slowl filled with warm solent% :ome ofthe desired compound will then dissole in the warm solent% When the :ohlet chamber is

almost full, the chamber is automaticall emptied b a siphon side arm, with the solent running

bac+ down to the distillation flas+% The thimble ensures that the rapid motion of the solent does

not transport an solid material to the still pot% This ccle ma be allowed to repeat man times,

oer hours or das% During each ccle, a portion of the non*olatile compound dissoles in the

solent% After / ccles of the Pet% ether,#" ccles of the 1thle acetate,## ccles of the Acetone

; #! ccles of the 8ethanol is concentrated in the distillation flas+% The adantage of this

sstem is that instead of man portions of warm solent being passed through the sample,


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i.4.!: So"/let a22arat$s

4.4. Sol#ent reco#er3 %3 $sin Rotar3 E#a2orator:

A rotar eaporator is a speciall designed instrument for the eaporation of solent

(single*stage or straight distillation) under acuum% The eaporator consists of a heating bath

with a rotating flas+, in which the li.uid is distributed as a thin film oer the hot wall surfaces

and can eaporate easil% The eaporation rate is regulated b the heating bath temperature, the

si>e of flas+, the pressure of distillation and the speed of rotation%


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i.4.0:Rotar3 e#a2orator

Ta%le.4.!: Partic$lars o sol#ent reco#er3 or o$r sol#ents

S.no E"tract oillin 2oint C/illin

te(2arat$re

R2(

# Petroleum ether

etract

-""*/"" c /"c !" rpm

! 1thl acetateetract

7-

"

*7/

"

c /

"

c !" rpm

& Acetone etract $-%$"c /" c !" rpm

' 8ethanolic

etract

-'%-" c /"c !" rpm


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4.5. PRELIMINAR6 PH6TOCHEMICAL SCREENIN7 O E8TRACTS:

Test sol$tion:Depending upon the tpe of natural drug under eamination, the test solutions,

pet% ether, ethl acetate, acetone and methanolic etracts were used%

Different tests were performed as follows?

1. Detection o Al9aloi's:

a+ Draen'ors test

To # m of test filtrate, two drops of Dragendorff@s reagent (Potassium bismuth iodide

solution) was added and obsered for the Formation of prominent reddish brown precipitate%

%+ Ma3ers test

# m of test filtrate was ta+en into a test tube and added two drops of 8aer@s reagent

(Potassium mercuric iodide solution) along the sides of the test tube and obsered for white or

cream precipitate%

c+ )aners test

# m of test filtrate was ta+en into a test tube, added two drops of Wagner@s reagent

(Iodine*Potassium iodide solution) along the sides of the test tube and obsered for reddish

brown precipitate%

'+ Haers test

To # m of filtrate, two drops of 5ager@s reagent (Picric acid) was added and obsered

for prominent ellow precipitate%

!. Detection o Car%o/3'rates:

a+ Molisc/s test

# m of the test solution was ta+en and two drops of alcoholic solution of B naphthol

(8olisch@s reagent) was added% The miture was sha+en and # m of conc% 5!:'was added

slowl from the sides of the test tube% The test tube were cooled in ice water and allowed to


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stand% Then the test tubes were obsered for iolet ring formation at the


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%+ Lie%er(ann ; $rc/ar's test

To the test filtrate, ! m of acetic anhdride, ! m of chloroform were added and heated

to boiling and cooled% Then # m of concentrated sulphuric acid was added along the sides of the

test tube and obsered for the formation of grass*green colour at the


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+ Test or sa2onin l3cosi'es

oa( test

Filtrates were ta+en and !" m of distilled water was added and sha+en for #$ min in a

graduated clinder and obsered for formation of a laer of stable foam&-, &7%

4.


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li+e cells grow in monolaers% rowth can also be inhibited using tumor necrosis factor alpha

(T9F alpha) (Ansari A. et al. *!B15+)%

MDA-M-!01 %reast cancer cell lines:

The 8DA*8*! breast cancer cell line was obtained from a patient in #67& at 8% D%

Anderson Cancer Center% With epithelial*li+e morpholog, the 8DA*8*! breast cancer cells

appear phenotpicall as spindle shaped cells% In itro, the 8DA*8*! cell line has an

inasie phenotpe% It has abundant actiit in both the oden chamber chemoinasion and

chemotais assa% The 8DA*8*! cell line is also able to grow on agarose, an indicator of

transformation and tumorigenicit, and displas a relatiel high colon forming efficienc '"% In

io, the 8DA*8*! cells form mammar fat pad tumors in nude mice% IG ine cells in growth medium supplemented with #"2 () D8: in # ml ali.uots of


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approimatel "%$*# #"-cells% When grown on #$ cm dishes, the cells reach confluence at K!%-

= #"-cells per dish%

Cell C$lt$re & Passae:

#% Thaw a #*ml ali.uot of cells as .uic+l as possible in water bath at &70C% Transfer cells to

!' m warm media in a $" m conical tube% 8i gentl% Plate the cells in a #$cm 9unc

delta surface plates% Place in incubator% After one da, remoe the medium and add fresh

media%

!% When cells are $"*-"2 confluent (meaning that er few of them are phsicall touching

each other), split #?' or #?$ (at most)% It is important to not let the cells become full

confluent because the can begin to fuse and partiall differentiate upon cell*cell contact%

To passage, remoe and discard culture medium% Einse twice with P: (Calcium and

8agnesium free)% For a cm dish, add !%$m of "%!$2 (w) trpsin H "%$& m8 1DTA

solution (ibco J!$&"") pre*warmed to &70C, and obsere cells under an inerted

microscope until cell aspect changes to round (usuall within -"*6" seconds)% Aspirate

the ma


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$% To detach the cells from the dishes, add dilute trpsin (!m P: H "%'m of ibco

trpsinH1DTA (ibco J!$&"")) for #" min at &70C, then .uench with #""u horse serum

or F:% Transfer to ice or '0C%

-% Add ! m of cold P: and scrape into a #$m falcon tubeM rinse plate once with $m of

cold P: and combine%

7% Pellet cells at &-" = g for $ minutes at '0C%

/% Aspirate P:trpsin solution and resuspend cells in $ ml cold ('0C) P: H # u8 P8:F%

6% Pellet cells at &-" = g for $ minutes at '0C%

#"% Carefull aspirate P: and add - ml cold ('0C) Farnham lsis buffer ($ m8 PIP1: p5

/%"

##% m8 NCl "%$2 9P*'") H Eoche Protease Inhibitor Coc+tail Tablet (Complete

##/&-#'$""#)% This step lses the cell membrane, leaing the nuclear enelope intact%

#!% Pellet nuclei at &-" = g for $ minutes at '0C%

#&% Place the nuclear pellet on ice% Carefull remoe supernatant and either proceed to

sonication step or snap free>e in li.uid nitrogen and store at */"0C or in li.uid nitrogen'#%

4.@.!. C$lt$re o MC-@ Cell lines:


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Cells are grown at &70C in a humidified incubator with $2 C!%

final :toc+ e(ample

D818 &6$ mF: (fetal boine serum) #"2 #""2 #"" m

Final $"" 8l

Materials:

D818 (high glucose H glutamine, no :odium Pruate)

F:

Antibiotics? We use #= Penicillin:treptomcin (#""= stoc+ 3 ibco J #$#'")% This comes out

to final concentrations of #"" unitsm penicillin and #"" ugm streptomcin%

Li$i' Nitroen Storae:

Free>e cells in growth medium supplemented with #"2 () D8: in # ml ali.uots of

approimatel "%$*# #"-cells% When grown on #$ cm dishes, the cells reach confluence at K!%-

= #"-cells per dish%

Proce'$re or C$lt$rin o MC-@ Cells:

#% Lsed 1agle@s 818, supplemented with #"2 F:, #2 penicillinstreptomcin% Can also

add non essential amino acids ("%# m8), Insulin (#"ugm) and :odium pruate

(#m8)% Add #"n8 estrogen to media for a &*' increase in cell numbers% 8aintain

temperature at &70C in humidified, concentrated C! ($2) atmosphere%

!% nce 8CF*7 cells reach approimatel 6"2 confluence on plates, remoe media and

passage cells b rinsing with #P: twice%

&% Add !*& m of warm (&70C) "%!$2 Trpsin* "%$& m8 1DTA solution to cells to disperse

cell laer% bsere under an inerted microscope% Dispersal should happen between $ and

#$ minutes% If cells are not detaching properl, place flas+ bac+ in &70C incubation

chamber% Do not incubate for more than & minutes or so% 9ote? Do not agitate the chills


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during dispersal, either b hitting or sha+ing the flas+% This ma cause clumping as the

cells detach%

'% nce 8CF*7 cell laer is dispersed (&min at &70C) deactiate Trpsin b adding $ ml

cellsTrpsin*1DTA to #"m of complete growth medium (see step #) in sterile tube%

Aspirate cells b gentl pipetting

$% Centrifuge cells in growth medium for $ minutes at #!$ g*force%

-% Eemoe trpsingrowth medium suspension from tube%

7% Eesuspend pellet (8CF*7 cells) in #" m fresh growth medium (see step #)

/% Plate # m of suspension to each new plate containing 6 m original growth medium (see

step #), and incubate at &70C in humidified $2 C! atmosphere%

[email protected]. C$lt$rin o MDA-M-!01 cells:

Me'i$( or MDA-M-!01:

#% C$lt$re Me'i$(:D*818 (high glucose), #"2 fetal boine serum (F:), "%# m8

818 9on*1ssential Amino Acids (91AA), ! m8 *glutamine, #2 Pen*:trep%

!% reee Me'i$(:6"2 F:, #"2 D8:%

Met/o's Esta%lis/in MDA-M-!01 C$lt$res ro( roen Cells:

#% Place #" m of complete D818 growth medium in a $"*m conical tube% Thaw the

fro>en croial of cells within #B! minutes b gentle agitation in a &70C water bath%

Decontaminate the croial b wiping the surface of the ial with 7"2 () ethanol%

!% Transfer the thawed cell suspension to the conical tube containing #" ml of growth

medium%

&% Collect the cells b centrifugation at #""" rpm for $ minutes at room temperature%

Eemoe the growth medium b aspiration%'% Eesuspend the cells in the conical tube in #$ m of fresh growth medium b gentl

pipetting up and down%

$% Transfer the #$ m of cell suspension to a T*7$ tissue culture flas+% Place the cells in a

&70C incubator at $2 C!%


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4.. MTT Assa3:

Princi2le:

This is a colorimetric assa that measures the reduction of ellow &*(',$*dimeththia>ol* !*l)*

!,$*diphenl tetra>olium bromide (8TT) b mitochondrial succinate dehdrogenase% The 8TT enters

the cells and passes into the mitochondria where it is reduced to an insoluble, coloured (dar+ purple)

forma>an product% The cells are then solubilised with an organic solent (eg% isopropanol) and the

released, solubilised forma>an reagent is measured spectrophotometricall% :ince reduction of 8TT can

onl occur in metabolicall actie cells the leel of actiit is a measure of the iabilit of the cells'&%

i.4.4: or(ation o or(aan

Materials:

P: 8TT ($ mgml in P:) B filter and +eep dar+, prepare freshl Acidic isopropanol ("%#9

5cl in absolute isopropanol) 6-*well plate (flat bottom)

Proce'$re:

#% Plate cells ($"""cellswell) in !"" ul P: in 6-*well (flat bottom)%!% The cells were treated with crude etracts ; with respectie controls for !'hrs,then #%$Ol!""ul

media was added%


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&% Add !" ul of 8TT solution, mi well%

'% Incubate for &h in &7C in dar+%

$% Eemoe the entire media ; 8TT solutionM add $" ul of D8: to the wells and mi well%-% Incubate additional #h in &7C in dar+%

@. Eead plate in 1I:A Eeader B measure D in $7"nm (bac+ground waelength is -&"nm) ''%

i.4.5: MTT ell 2late

4..1. IC5BDeter(ination:

IC$"is the acronm for half maimal inhibitor concentrationQ% IC$"alue indicates the

concentration needed to inhibit a biological or biochemical function b half (e%g% inhibition of en>mes,

affinit to cell receptors)% In pharmaceutical research, it is a fre.uentl used unit to specif the in itro

potenc of a drug or a 9C1%

The actiit of an en>me is determined after eposure to a series of inhibitor concentrations%

IC$"is calculated b the following formula?


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IC5BF *5B,-LoIn/,+G*Hi/In/,-LoIn/,+"*Hi/Conc-LoConc+ LoConc

LoIn/,G Hi/In/,: , in/i%ition 'irectl3 %elo G a%o#e 5B, in/i%ition

LoConcG Hi/Conc: Corres2on'in concentrations o test co(2o$n'

4..!. Har#estin Cells >se' or Assa3:

#% Cells used for bioassa are tpicall from stoc+ culturesM howeer, the culture conditions used to

grow cells can affect results% We recommend that culture conditions be ta+en into consideration

when anal>ing results of proliferation bioassas% Eecord the following culture conditions? passage

number, medium composition, cell densit and time in culture since last medium change%

!% Wash the cells twice b centrifugation in assa medium that is free of the growth factor(s) to be

tested%

&% Determine cell number and trpan blue iabilit, and suspend the cells to a final concentration of #

4 #"$ml in assa medium%

'% Dispense $"Ol of the cell suspension ($,""" cells) into all wells of the pre*e.uilibrated 6-*well

plate (:ection III%A, :tep !)% The total olume in the plate should now be #""Olwell%$% Incubate the plate at &70C for '/B7! hours in a humidified, $2 C! atmosphere''%

4.?. Protein esti(ation:

Proteins are polmers of amino acids% Twent different tpes of amino acids occur naturall in

proteins% Proteins differ from each other according to the tpe, number and se.uence of amino acids

that ma+e up the polpeptide bac+bone% As a result the hae different molecular structures, nutritional

attributes and phsiochemical properties% Proteins are important constituents of foods for a number of

different reasons% The are a mae%

4.?.1. ESTIMATION O PROTEINS 6 RADORD METHOD:


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Princi2le

The protein in solution can be measured .uantitatiel b different methods% The methods

described b radford uses a different concept*the proteinRs capacit to bind to a de, .uantitatiel%

The assa is based on the abilit of proteins to bind to coomassie brilliant blue and form a comple

whose etinction coefficient is much greater than that of free de'$%

List o Reaents an' Instr$(ents

E$i2(ent

o Test tubes

o raduated clinder

o Weight alance

o LG spectrophotometer

Reaents

Dissole #""mg of Coomassie*rilliant blue !$" in $" ml of 6$2 1thanol%

Add #"" ml of /$2 phosphoric acid and ma+e up to -"" ml with distilled water%

Filter the solution and add #"" ml of glcerol, then ma+e upto #"""ml%

The solution can be used after !' hrs%

:A

Proce'$re:

Prepare arious concentration of standard protein solutions from the stoc+ solution ( "%!, "%',

"%-, "%/ and #%"ul ) into series of test tubes and ma+e up the olume to #!$ u %


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Pipette out !ul of the sample in 6-*well plate and ma+e up the olume to #!$u%

A tube with #!$ul of controles seres as blan+

Add #!$ ul of coomassie brilliant blue to each tube and mi b orte or inersion%

Wait for #"*&"minutes and read each of the standards and each of the samples at $6$nm%

Plot the absorbance of the standards erses their concentration%

Plot graph of optical densit ersus concentration% From graph find amount of protein in

un+nown sample'-,'7%

4.1B. SDS-PA7E:

:D:*PA1 is widel used to anal>e the proteins in comple etracts% The most

commonl used methods are deried from the discontinuous :D:*PA1 sstem first described

b aemmli (#67")% The sstem actuall consists of two gels * a resoling (a+a running) gel in

which proteins are resoled on the basis of their molecular weights (8Ws) and a stac+ing gel in

which proteins are concentrated prior to entering the resoling gel% Differences in the

compositions of the stac+ing gel, resoling gel and electrophoresis buffer produce a sstem that

is capable of finel resoling proteins according to their 8Ws%

7el electro2/oresis o (acro(olec$les

In gel electrophoresis, an electric field is used to moe charged molecules through

a matri of a polmeri>ed substance such as agarose or polacrlamide% The rates at which

indiidual molecules moe through the gel depend on the properties of both the separation

sstem and the molecules themseles% el matrices are permeated with networ+s of pores

through which the molecules moe% The amount of resistance that the matri presents to

the moement of a molecule depends on the diameter of the pore as well as the si>e and

geometr of the molecule% Eesearchers can control the si>e of the pore b ade of a


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linear D9A molecule can be estimated from the rate at which it moes through an agarose gel,

because D9A molecules hae a uniform charge to mass ratio% Protein electrophoresis is

somewhat more complicated than D9A electrophoresis% Proteins are much smaller than D9A

molecules, so polacrlamide gels are used for their separation% In addition, proteins are much

more structurall dierse than D9A, so chemical treatments are used to impart a uniform

geometr and chargemass ratio to the proteins%

C/e(istr3 o acr3la(i'e 2ol3(eriation

The polacrlamide gels used to separate proteins are formed b the chemical

polmeri>ation of acrlamide and a cross*lin+ing reagent, 9,9@methlenebisacrlamide

(opposite page)% Inestigators are able to control the si>e of the pores in the gel b ad


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constant, will decrease the pore si>e of the gel% Polmeri>ation occurs because of free ogen

radicals that react with the inl groups in acrlamide and bisacrlamide, as shown in the figure

below% The ogen radicals are generated from the catalst, ammonium persulfate (AP:), when

it reacts with a second catalst, 9,9,9@,9@*tetramethlethlenediamine (T181D)%

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Proteins are 'enat$re' 2rior to electro2/oresis

Compared to D9A molecules, proteins are structurall er dierse% Proteins show

tremendous ariation in their amino acid compositions and in the distribution of amino acids

in their folded structures, features with important implications for electrophoresis% Eecall that

proteins are mitures of hdrophobic and hdrophilic amino acids and that the primar se.uence

of the protein determines its final folded form% ecause of the hdrophobic effect, the surfaces of

proteins proteins hae a higher fre.uenc of polar and charged amino acids than the interiors,

where hdrophobic residues predominate% Folded proteins assume man different geometries

and their surfaces are mosaics with respect to the distribution of E groups with different

chemistries% ecause proteins are so dierse with respect to their surface charges and geometries,

the molecular weights offoldedproteins cannot be simpl determined b their migration rate in

an electric field% Postiel and negatiel charged proteins would migrate in different directions%

To resole the proteins in a sample according to their si>e, inestigators must conert the

proteins to a uniform geometr and impart a uniform chargemass ratio to the proteins% In :D:*

PA1, the solution is to denature the proteins b boiling them with the anionic detergent, sodium

dodecl sulfate (:D:) and !*mercaptoethanol% The combination of heat and detergent is sufficient

to brea+ the man non*coalent bonds that stabili>e protein folds, and !*mercaptoethanol brea+s

an coalent bonds between csteine residues% i+e other detergents, :D: is an amphipathic

molecule, consisting of a hdrophobic #!*carbon chain and a hdrophilic sulfate group% The :D:

hdrocarbon chain permeates the protein interior and binds to hdrophobic groups, reducing the

protein to a random

Proteins are #is$alie' it/ stains

With few eceptions, naturall*occurring proteins are inisible on :D:*PA1 gels%

Conse.uentl, researchers often use pre*stained protein standards to monitor the approimate

positions of proteins during electrophoresis% The pre*stained standards are produced b

coalentl attaching a large number of chromophores to a protein% The addition of the

chromophores increases the 8W of the protein and also produces more diffuse bands on the gel%

The diffuseness of the bands reflects ariation in the number of de molecules attached to


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indiidual protein molecules% We will use prestained standard proteins in our gels, so ou will

be able to isuali>e the protein separation that is occurring% Seast proteins will not be isible,

howeer, because the hae not been modified with chromophores%

To isuali>e the positions of proteins after electrophoresis is complete, inestigators

stain the gels with arious des that bind noncoalentl and with er little specificit to

proteins% During the staining process, proteins are also fiedQ in the gel, meaning that proteins

become insoluble and unable to diffuse out of the gel% In our eperiments, we will use a

colloidal suspension of rilliant lue *!$", also +nown as Coomassie lue % rilliant lue

*!$" binds proteins nonspecificall through a large number of ionic and Gan der Waals

interactions% In this procedure, gels are rinsed with water to remoe the buffer salts used for

electrophoresis and then treated with the colloidal *!$" suspension% Protein bands appear

rapidl, and when necessar, the gels can be destained with deioni>ed water to lower the gel

bac+ground% rilliant lue staining intensit is considered to be a .uantitatie procedure,

because with some eceptions, the intensit of a stained band is directl proportional to the

amount of protein in a band%

Protein (olec$lar ei/ts can %e calc$late' ro( t/eir (iration on els

The si>es of proteins in an etract can be calculated b comparing their migration to a set

of standard proteins run on the same gel%

The principles used to estimate 8Ws are the same used for agarose gel electrophoresis% A plot of

the log#"8W of the standard proteins against the distance that each protein migrated on the gel

will gie a straight line in the region where the gel has good resoling power% The si>es of

un+nown proteins can be estimated b interpolating eperimental alues on a graph of standard

proteins% Proteins whose molecular weights fall outside this range will not be well*resoled on

the gel%


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Pre2are to resol#in els:

The table below shows the .uantities of each chemical that will need to pour two

gels with the 8ini*Protean sstem% Polmeri>ation occurs rapidl, so be sure to follow the

step*b*step instructions below%

or 1B, Resol#in 7el:

Ta%le.4.0: Resol#in el stoc9in els reaents & t/eir $antities.

Reaent Resol#in el *1B,+

-or ! els*15(l+ or 1 el *(l+

Deioni>ed water $%6 m &%#$ m

&"2 acrlamide?bis*acrlamide (!6?#) $%" m !%-7 8l

#%$ 8 Tris*5Cl, p5 /%/ &%/ m !%" m

:D: #"2 "%#$ m "%"/ 8l

#"2 ammonium persulfate (catalst) "%#$ m "%"/ m

T181D (catalst) "%""- m, "%""&! m

or 5, Stac9in 7el:

Reaent

Stoc9in el *5,+

-or ! els*ed water '%# m &%#$ m

&"2 acrlamide?bis*acrlamide (!6?#) #%" m !%-7 m

#%$ 8 Tris*5Cl, p5 /%/ "%7$ m !%" m

:D: #"2 "%"- m "%"/ m

#"2 ammonium persulfate (catalst) "%"- m "%"/ m

T181D (catalst) "%""- m, "%""& lm

R$nnin %$er 18 *!L+:


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Ta%le.4.4: Reaents & t/eir $antities or 2re2aration o 18 r$nnin %$er.

Inre'ient $antit3

lcine !/%/ g

Tries base -%"'g:D: !%"g

radford method for estimating proteins was done and &"ug of the concentration was loaded

into the wells%

Proce'$re:

#% 5eat the sample at 6$

"

C for $ min, ta+e the sample out spin them down% Place thegel plate such that smaller plate faces inside of tan+%

!% oad all samples ;added mar+er 'Ol%

&% Eun the gel at -"G for about !hrs (nce the samples cross trac+ing gel increase

the oltage to /"G)%

'% Eemoe the gel ; assemble the blot for transfer b wetting the blot ;

membrane in transfer buffer %

$% Place the blot ; run at &" G oer night or #""G for ! hrs (with ice pac+sinside)%

-% Eemoe the membrane ; add ponceau stain%

7% Wash it with T:T till the stain completel remoed%

/% loc+ it with $2 mil+ for thaw%6% Add #" Antibod (Eun =!) (#?!""" dilution) ta+e $Ol anti bod in #" ml of

#"% $2 mil+ in T:T ; add #" Ol of sodium a>ide%

##% Incubated it oer night at '"C% Then remoe #"anti bod%

#!% ##% Wash with T:T &*$ times (#st time for #" min, !ndtime for $ min, &rd time

for $ min)%

#&% #!% Add #" ml of !" anti bod ; Incubate for !hrs at ET (! ml of $2 mil+ and

T:T)%

#'% Wash with T:T for & times (#st time for #" mins, !ndtime for $ mins, &rd time

for $ mins)%#$% the blot was deeloped in Chemi Doc =E:H :stem with Image ab

:oftware

Stri22in %$er:

Ta%le.4.5: Reaents & t/eir $antities or t/e 2re2aration o stri22in %$er .

The following reagents are re.uired to prepare striping buffer,

Reaents or 1BB (l or !B (l


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:D:(#"2) !" ml ' ml

"%$8 Tris 5cl #!%$ ml !%$ ml

Lltrapure 5! -7%$ ml #&%$ml

U* 8ercapto ethanol "%/ ml "%#-ml

Proce'$re:

#% Warm stripping buffer to $$"C, then add the buffer to the membrane and incubated

for '$ min at $$" C %

!% Einse the blot with water for # hrs%

&% Wash it with T:T $ times ($ mins each) change the buffer ;also bo%

'% loc+ with mil+ for # hrs%$% Incubate oer night with #" anti bod at '"C%

-% Eemoe #" anti bod ; wash it with T:T $ times (each $ mins)%loc+ with mil+ for

# hrs or ET%

7% Add !" anti bod ; incubate it oer night at '"C '/%

4.11. )ESTERN LOTTIN7:

Western lotting was performed b the rapid method of Towbin et al%, (#676) to

detect the epression pattern of a protein% To detect the antigens blotted on a nitrocellulose

membrane with the use of an antibod%

PRINCIPLE:

Western blotting (also +nown as protein blotting or immunoblotting) is a rapid and

sensitie assa for detectie and characteri>ation of proteins% Western blotting techni.ue


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eploits the inherent specificit b polclonal or monoclonal antibodies%

It is an analtical method wherein a protein sample is electrophoresed on an :D:*

PA1 and electrotransferred onto nitrocellulose membrane% The transferred protein is

detected using specific primar antibod and secondar en>me labeled antibod and

substrate% A protein sample is subme which, when proided with a

chromogenic substrate, will cause a color reaction% Thus the molecular weight and amount of

the desired protein can be characteri>ed from a comple miture (e%g% crude cell etract) of

other proteins b western blotting'6%

REA7ENTS AND MATERIALS:

a% 9itrocellulose membrane

b% Plastic staining bo

c% 1lectroblotting apparatus

d% Whatmann 9o%# filter paper

Transer %$er *5BB (l 2H.0+:

TrisB5Cl *!$ m8

lcine *#6! m8 8ethanol*!"2

1B8 Tris %$ere' saline *TS+ *1BB (l 2H @.


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loc9in sol$tion *5B(L+:

$2 9on*dr fat mil+ powder * "%!$g

#= T: (p5 7%-) * $"m

"%#2 Tween* !" * "%"$m

)as/in %$er *1BB(L+:

(T:) #= T: (p5 7%-) *#"" ml "%#2 Tween * !" * "%# ml

"%"$2 "f &@&* diaminoben>idine tetra hdrochloride (DA) substrate and "%"#2 of

5!! were dissoled in #= P: (p5 7%-)% This chromogen substrate was prepared


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PROCED>RE:

#% After :D:*PA1, the gel was e.uilibrated in blotting buffer for !" min at roomtemperature% While the gel was e.uilibrating, a piece of nitrocellulose membrane was cut

into the same dimension as the gel it was wet slowl b sliding it at '$o angle into

transfer buffer and was soa+ed for !" min%

!% The pieces of Whatmann 9o%# filter paper, four pads were also soa+ed in transfer buffer

for !" min%

&% Then, the pads, filter paper, nitrocellulose membrane and gel were assembled in the semi*

dr blot apparatus in the following order?

The two presoa+ed pads were placed at the bottom and a glass pipette was rolled oer

the surface of the pad to remoe air bubbles% Then, the Whatmann 9o%# filter paper

was placed followed the nitrocellulose membrane% Carefull, the e.uilibrated gel was

placed on top of the nitrocellulose membrane% The second Whatmann 9o%# filter

paper and followed it, the second set of pad were placed on top of the gel% (After each

step care was ta+en to remoe the bubbles)% The transfer cell and plug was assembled

and the gel transferred for !h at !$G#&"%

After the transfer, protein were isuali>ed b staining in ponceau : solution for $ min,

destained in the distilled water and the molecular mar+er was mar+ed with in deliblein+ and destained for #" min% The membrane was bloc+ed in bloc+ing buffer for #h at

room temperature% Then, the membrane was washed again with washing buffer and

incubated with primar antibod oernight at 'oC% The net da, the membrane was

washed again with washing buffer and incubated with 5EP*con


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i.4.@: Processes o transer o 2rotein ro( el to nitrocell$lose (e(%rane.

i.4.: Sc/e(atic 'iara( o )/ole 2rocess o SDS-PA7E &)estern %lottin.

Documents

Materials and Methods Final