S K Patel College Of Pharmaceutical Education & Research

Ganpat UniversityGanpat University

((((

NorthernNorthern blotblot for RNARNANorthernNorthern blotblot for RNARNA

Western blotWestern blotWestern blotWestern blotWestern blotWestern blot

for ProteinsProteins. for ProteinsProteins.

BY,BY,

Nirmal PatelNirmal PatelSouthern blotSouthern blot for DNADNA.

M Pharm (Bio Tech)Southern blotSouthern blot for DNADNA.

M Pharm (Bio Tech)

The Western blot is an aanalyticalnalyticalThe Western blot is an aanalyticalnalyticalThe Western blot is an aanalyticalnalyticalThe Western blot is an aanalyticalnalytical

techniquetechnique used to detect specific techniquetechnique used to detect specific techniquetechnique used to detect specific techniquetechnique used to detect specific

proteinsproteins in a given sample of proteinsproteins in a given sample of proteinsproteins in a given sample of

tissue homogenate or extract.tissue homogenate or extract.tissue homogenate or extract.tissue homogenate or extract.

-može da se koristi za dokazivanje At ili Ag-može da se koristi za dokazivanje At ili Ag

-osetljiva i specifična tehnika (ELISA - specifičnija)-osetljiva i specifična tehnika (ELISA - specifičnija)-osetljiva i specifična tehnika (ELISA - specifičnija)

The name Western blot was given to the technique by W. Neal Burnette.The name Western blot was given to the technique by W. Neal Burnette.

WBWBWBIt uses gel electrophoresis to separate

WBIt uses gel electrophoresis to separate It uses gel electrophoresis to separate

native or denatured proteinsnative or denatured proteinsnative or denatured proteins

by the length of the polypeptide by the 3-D structure of the protein by the length of the polypeptide

(denaturing conditions)

by the 3-D structure of the protein

(native conditions) (denaturing conditions)(native conditions)

Contents:Contents:Contents:Contents:Contents:Contents:Contents:Contents:Steps in a Western blot:Steps in a Western blot:

- Tissue preparation - Tissue preparation

- Gel electrophoresis- Gel electrophoresis

- Transfer- Transfer- Transfer

- Blocking - Blocking - Blocking

- Detection - Two step detection- Detection - Two step detection

- One step detection- One step detection

Analysis- Analysis - Colorimetric detection- Analysis - Colorimetric detection

- Chemiluminescent detection- Chemiluminescent detection

- Radioactive detection- Radioactive detection- Radioactive detection

- Fluorescent detection- Fluorescent detection

Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:Tissue preparation:- Samples may be taken from - Samples may be taken from

-- whole tissuewhole tissue, from cell culturecell culture-- whole tissuewhole tissue, from cell culturecell culture-- whole tissuewhole tissue, from cell culturecell culture

-- bacteriabacteria, virusvirus or -- bacteriabacteria, virusvirus or -- bacteriabacteria, virusvirus or

-- environmental samplesenvironmental samples.-- environmental samplesenvironmental samples.-- environmental samplesenvironmental samples.Cells may also be broken open

-- firstfirst broken down mechanicallymechanically

Cells may also be broken open

by one of the mechanical methods.-- firstfirst broken down mechanicallymechanically

by one of the mechanical methods.-- firstfirst broken down mechanicallymechanically

blenderblender (for larger sample volumes),blenderblender (for larger sample volumes),

homogenizerhomogenizer (smaller volumes), or homogenizerhomogenizer (smaller volumes), or homogenizerhomogenizer (smaller volumes), or

by sonicationsonication..by sonicationsonication..

detergents, salts, and buffersdetergents, salts, and buffers

{ }-- BiochemicaBiochemical l techniquesdetergents, salts, and buffersdetergents, salts, and buffers

{ }-- BiochemicaBiochemical l techniquesAnti Anti Protease and Protease and phosphatasephosphatase{ }Anti Anti Protease and Protease and phosphatasephosphatase{ }

-- mechanicalmechanical techniques - various types of filtrationfiltration and centrifugationcentrifugation.-- mechanicalmechanical techniques - various types of filtrationfiltration and centrifugationcentrifugation.

- to encourage lysislysis of cells and to solubilize proteinssolubilize proteins, - to encourage lysislysis of cells and to solubilize proteinssolubilize proteins, - to encourage lysislysis of cells and to solubilize proteinssolubilize proteins,

may be employed :may be employed :may be employed :

detergents, salts, and buffersdetergents, salts, and buffersdetergents, salts, and buffersdetergents, salts, and buffersdetergents, salts, and buffersdetergents, salts, and buffers

SDS - Sodium Dodecyl Sulfate (a detergent a detergent used in many western blotting solutions) SDS - Sodium Dodecyl Sulfate (a detergent a detergent used in many western blotting solutions)

digestiondigestion- to prevent the digestiondigestion of the sample by its own enzymes :- to prevent the digestiondigestion of the sample by its own enzymes :

Anti Anti Protease and Protease and phosphatasephosphataseAnti Anti Protease and Protease and phosphatasephosphataseAnti Anti Protease and Protease and phosphatasephosphatase

denaturingdenaturing- When need to avoid protein denaturingdenaturing- When need to avoid protein denaturingdenaturing

cold temperaturescold temperaturesttissue preparation is often done at issue preparation is often done at cold temperaturescold temperaturesttissue preparation is often done at issue preparation is often done at cold temperaturescold temperatures

Tissue PreparationTissue PreparationTissue PreparationTissue Preparation

Rastvori za denaturaciju sadrže -HClRastvori za denaturaciju sadrže -HCl

-SDS-SDS

-Merkaptoetanol-MerkaptoetanolKoriste se za vizuelizaciju kretanja

-Bromfenol plavo →→Koriste se za vizuelizaciju kretanja

proteina kroz gel tokom izvođenja -Bromfenol plavo →→

--PyroninPyronin →→proteina kroz gel tokom izvođenja

elektroforeze.--PyroninPyronin →→

-Ureuelektroforeze.

-Ureu

-Glicerol-Glicerol

Gel electrophoresis:Gel electrophoresis:Gel electrophoresis:Gel electrophoresis:Gel electrophoresis:Gel electrophoresis:Gel electrophoresis:Gel electrophoresis:- The proteins are separated using gel electrophoresisgel electrophoresis..- The proteins are separated using gel electrophoresisgel electrophoresis..- The proteins are separated using gel electrophoresisgel electrophoresis..

- Separation of proteins may be done by - Separation of proteins may be done by

isoelectricisoelectric point (point (pIpI)) - - - IEF razdvajanje isoelectricisoelectric point (point (pIpI)) - - - IEF razdvajanje (pH 3.5-10)isoelectricisoelectric point (point (pIpI)) - - - IEF razdvajanje (pH 3.5-10)

molecular weightmolecular weight * * -- -- -- SDS razdvajanjeSDS razdvajanjemolecular weightmolecular weight * * -- -- -- SDS razdvajanjeSDS razdvajanjemolecular weightmolecular weight * * -- -- -- SDS razdvajanjeSDS razdvajanje

electric chargeelectric charge, or electric chargeelectric charge, or electric chargeelectric charge, or

a combination of these factors.a combination of these factors.a combination of these factors.

- most common type of gel electrophoresis - most common type of gel electrophoresis - most common type of gel electrophoresis

SDSSDS--PAGE (SDS polyacrylamide gel electrophoresis) PAGE (SDS polyacrylamide gel electrophoresis) SDSSDS--PAGE (SDS polyacrylamide gel electrophoresis) PAGE (SDS polyacrylamide gel electrophoresis) SDSSDS--PAGE (SDS polyacrylamide gel electrophoresis) PAGE (SDS polyacrylamide gel electrophoresis)

-- maintains polypeptides in a denatureddenatured statestate, thus allows-- maintains polypeptides in a denatureddenatured statestate, thus allows-- maintains polypeptides in a denatureddenatured statestate, thus allows

separation of proteins by their *molecular weightmolecular weight tjtj po broju AKpo broju AK. separation of proteins by their *molecular weightmolecular weight tjtj po broju AKpo broju AK.

Priprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gela

- Vinil polimerizacija monomera akrilamida (AA) u - Vinil polimerizacija monomera akrilamida (AA) u - Vinil polimerizacija monomera akrilamida (AA) u

duge lance poliakrilamida koji su povezani manjim duge lance poliakrilamida koji su povezani manjim duge lance poliakrilamida koji su povezani manjim

količinama komonomera NN’-metilen-bis-količinama komonomera NN’-metilen-bis-količinama komonomera NN’-metilen-bis-

akrilamida (BIS A)akrilamida (BIS A) (NH(NH44))22SS22OO88akrilamida (BIS A)

- Katalizator polimerizacije je – amonijum persulfat amonijum persulfat

(NH(NH44))22SS22OO88

- Katalizator polimerizacije je – amonijum persulfat amonijum persulfat - Katalizator polimerizacije je – amonijum persulfat amonijum persulfat (za dejstvo je potrebna bazna sredina pa se dodaje baza TEMEDbaza TEMED)(za dejstvo je potrebna bazna sredina pa se dodaje baza TEMEDbaza TEMED)(za dejstvo je potrebna bazna sredina pa se dodaje baza TEMEDbaza TEMED)

-- Rezultat Rezultat –– gel mrežaste strukturegel mrežaste strukture-- Rezultat Rezultat –– gel mrežaste strukturegel mrežaste strukture-- Rezultat Rezultat –– gel mrežaste strukturegel mrežaste strukture

- Veličina pora se reguliše koncentracijama AA i BIS A- Veličina pora se reguliše koncentracijama AA i BIS A- Veličina pora se reguliše koncentracijama AA i BIS A

Priprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gelaPriprema gela• Parametri• Parametri• Parametri

T%T% • Masa AA i BIS A u 100ml rastvora gela [gr/ml]T%T% • Masa AA i BIS A u 100ml rastvora gela [gr/ml]T%T% • Masa AA i BIS A u 100ml rastvora gela [gr/ml]

CC%% • Odnos mase BIS A u odnosu na ukupnu masu AA i BIS ACC%% • Odnos mase BIS A u odnosu na ukupnu masu AA i BIS ACC%%CC%%

• Voditi računa o puferupuferu:• Voditi računa o puferupuferu:• Voditi računa o puferupuferu:

– pH– pH brzinubrzinu i rezoluciju i rezoluciju – pHUtiču nabrzinubrzinu i rezoluciju i rezoluciju

– MolarnostiUtiču nabrzinubrzinu i rezoluciju i rezoluciju

razdvajanjarazdvajanja– Molarnosti razdvajanjarazdvajanja• Sobna temperatura

razdvajanjarazdvajanja• Sobna temperatura

proteina• Sobna temperatura

proteina

****The concentration of acrylamideacrylamide determines the resolution of the gel.resolution of the gel.**The concentration of acrylamideacrylamide determines the resolution of the gel.resolution of the gel.

- the greatergreater the acrylamide concentration- the greatergreater the acrylamide concentration

the better the resolution of lower molecular weight proteinslower molecular weight proteins. the better the resolution of lower molecular weight proteinslower molecular weight proteins. the better the resolution of lower molecular weight proteinslower molecular weight proteins.

-The lowerlower the acrylamide concentration-The lowerlower the acrylamide concentration

the better the resolution of higher molecular weight proteinshigher molecular weight proteins. the better the resolution of higher molecular weight proteinshigher molecular weight proteins. the better the resolution of higher molecular weight proteinshigher molecular weight proteins.

** Samples are loaded into wellswells in the gel.** Samples are loaded into wellswells in the gel.**

** markermarker** One lane is usually reserved for a markermarker or or ladderladder, (mixture of proteins having (mixture of proteins having ** One lane is usually reserved for a markermarker or or ladderladder, (mixture of proteins having (mixture of proteins having

defined molecular weightsdefined molecular weights, typically , typically stainedstained so as to form visible, so as to form visible, colouredcoloured bands).bands).defined molecular weightsdefined molecular weights, typically , typically stainedstained so as to form visible, so as to form visible, colouredcoloured bands).bands).

** When voltage is applied along the gel, ** When voltage is applied along the gel, **

proteins migrate into it at different speeds. “Apply 15 µL of the proteins migrate into it at different speeds. “Apply 15 µL of the

molecular weight standards (different (different electrophoreticelectrophoretic mobilitiesmobilities).). molecular weight standards (different (different electrophoreticelectrophoretic mobilitiesmobilities).). molecular weight standards

to one or two wells...”to one or two wells...”

Polyacrylamide GelPolyacrylamide GelPolyacrylamide GelPolyacrylamide GelPolymerized gel:Polymerized gel:

--Resolving gels made in Resolving gels made in aka “preparativni gel” – se ne boji jer boja

--Resolving gels made in Resolving gels made in aka “preparativni gel” – se ne boji jer boja

može uticati na elekroforetska svojstva6%, 10%, 12%, 18%.6%, 10%, 12%, 18%.

može uticati na elekroforetska svojstva

ispitivanih proteina.6%, 10%, 12%, 18%.6%, 10%, 12%, 18%.

--Stacking Gel up to 5% Stacking Gel up to 5%

ispitivanih proteina.

--Stacking Gel up to 5% Stacking Gel up to 5%

was added to gel and then thewas added to gel and then the wells arewells are created.created.was added to gel and then thewas added to gel and then the wells arewells are created.created.

The percentage chosen The percentage chosen The percentage chosen

depends on the size of thedepends on the size of the proteinproteindepends on the size of thedepends on the size of the proteinprotein

that one wishes to identify (detect or that one wishes to identify (detect or that one wishes to identify (detect or

probe) in the sample. probe) in the sample.

The smaller it is the bigger the percentageThe smaller it is the bigger the percentage

Najčešće se koristi VERTIKALNA Najčešće se koristi VERTIKALNA Najčešće se koristi VERTIKALNA Najčešće se koristi VERTIKALNA

elektroforezaelektroforezaelektroforezaelektroforeza

Postoji: 1. gel za koncentrovanjePostoji: 1. gel za koncentrovanje

2. gel za separaciju2. gel za separaciju

Bazenčić se puni sa tzv running buffer (SDS, Glycine, baza), a zatim se dodaje Bazenčić se puni sa tzv running buffer (SDS, Glycine, baza), a zatim se dodaje

ispitivani protein rastvoren u denaturing buffer-uispitivani protein rastvoren u denaturing buffer-u

1. Gel za koncentrovanje1. Gel za koncentrovanje1. Gel za koncentrovanje1. Gel za koncentrovanje-blago kiseo (pH 6.5)-blago kiseo (pH 6.5)-blago kiseo (pH 6.5)

-niska koncentracija AA (5%) → porozan-niska koncentracija AA (5%) → porozan

-proteini se slabo razdvajaju ali su trake (linije) -proteini se slabo razdvajaju ali su trake (linije)

jasno definisanejasno definisane

2. Gel za separaciju2. Gel za separaciju2. Gel za separaciju2. Gel za separaciju- bazan (pH 8.8)- bazan (pH 8.8)

- viša koncent. AA (6-18%) → kanali (pore) su uži- viša koncent. AA (6-18%) → kanali (pore) su uži- viša koncent. AA (6-18%) → kanali (pore) su uži

-Proteini koncentrovani u gornjem gelu, prolaze -Proteini koncentrovani u gornjem gelu, prolaze

kroz uske pore separatnog gela koji omogućava kroz uske pore separatnog gela koji omogućava

lakše i brže kretanje manjim proteinima.lakše i brže kretanje manjim proteinima.

Bojenje gela se izvodi nakonnakon izvođenja WB Bojenje gela se izvodi nakonnakon izvođenja WB

da bi bili sigurni da je protein prešao na membranu!da bi bili sigurni da je protein prešao na membranu!(razlikovati bojenje membrane!!!)(razlikovati bojenje membrane!!!)

WB se izvodi u 3 faze:WB se izvodi u 3 faze:WB se izvodi u 3 faze:WB se izvodi u 3 faze:1. Ag se elektroforezom u poliakrilamidnom gelu rastavi na 1. Ag se elektroforezom u poliakrilamidnom gelu rastavi na 1. Ag se elektroforezom u poliakrilamidnom gelu rastavi na

sastavne delove koji se u gelu vide kao zasebne linije.sastavne delove koji se u gelu vide kao zasebne linije.sastavne delove koji se u gelu vide kao zasebne linije.

2. Gel se prekriva nitroceluloznom (ili dr.) membranom i 2. Gel se prekriva nitroceluloznom (ili dr.) membranom i 2. Gel se prekriva nitroceluloznom (ili dr.) membranom i

stavlja između dva filtera natopljena rastvorom pufera. stavlja između dva filtera natopljena rastvorom pufera. stavlja između dva filtera natopljena rastvorom pufera.

Proteini se prenose sa gela na membranu.Proteini se prenose sa gela na membranu.Proteini se prenose sa gela na membranu.

3. Membrana se uranja u rastvor At za pojedine Ag koja su 3. Membrana se uranja u rastvor At za pojedine Ag koja su

označena nekim enzimom. Nakon uranjanja i inkubacije, označena nekim enzimom. Nakon uranjanja i inkubacije, označena nekim enzimom. Nakon uranjanja i inkubacije,

trake se ispiraju da bi se uklonila nevezana At. Trake se trake se ispiraju da bi se uklonila nevezana At. Trake se trake se ispiraju da bi se uklonila nevezana At. Trake se

zatim uranjaju u rastvor supstrata. Supstrat se razgrađuje zatim uranjaju u rastvor supstrata. Supstrat se razgrađuje zatim uranjaju u rastvor supstrata. Supstrat se razgrađuje

i boji linije u kome su konjugovana At reagovala sa Ag.i boji linije u kome su konjugovana At reagovala sa Ag.i boji linije u kome su konjugovana At reagovala sa Ag.

Loading the SDSLoading the SDS--PAGE gelPAGE gelLoading the SDSLoading the SDS--PAGE gelPAGE gelLoading the SDSLoading the SDS--PAGE gelPAGE gelLoading the SDSLoading the SDS--PAGE gelPAGE gelPolyacrylamide Gel electrophoresiselectrophoresis..Sodium Dodecyl Sulfate Polyacrylamide Gel electrophoresiselectrophoresis..Sodium Dodecyl Sulfate

-SDS linearizes all proteins and gives net negative charge-SDS linearizes all proteins and gives net negative charge-SDS linearizes all proteins and gives net negative charge

dolazi do razdvajanja proteina po molekulskoj težinidolazi do razdvajanja proteina po molekulskoj težini

SDS - Sodium Dodecyl Sulfate SDS - Sodium Dodecyl Sulfate

(a detergent used in many (a detergent used in many

western blotting solutions) western blotting solutions)

(pore)(pore)

Veličina pora zavisi od koncentracijeVeličina pora zavisi od koncentracije

akrilamida (AA) i bis-akrilamida (BIS A)akrilamida (AA) i bis-akrilamida (BIS A)

-The Pink Strands are the Denatured ProteinsDenatured Proteins. See varying sizes-The Pink Strands are the Denatured ProteinsDenatured Proteins. See varying sizes-The Pink Strands are the Denatured ProteinsDenatured Proteins. See varying sizes

++ ––-They Are traveling to the ++ since they have –– charge due to SDS.-They Are traveling to the ++ since they have –– charge due to SDS.- - - - - - - - - - - - - - - -

++ ––- - - - - - - - - - - - - - - -

seperated by

- - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - -

-These strands go throught the tunnel and are seperated by - - - - - - - - - - - - - - - -

-These strands go throught the tunnel and are seperated by their their their

sizesize!!!!!!sizesize!!!!!! tj. tj. sizesize!!!!!! tj. tj. sizesize!!!!!! tj. tj.

molekulske težine molekulske težine molekulske težine molekulske težine molekulske težine molekulske težine

Denatured Denatured Denatured Denatured Denatured Denatured

ProteinsProteinsProteinsProteins

Proteins here are actually linearized.Proteins here are actually linearized.Proteins here are actually linearized.Proteins here are actually linearized.

Boja:Boja:Boja:

--Bromphenol blueBromphenol blue--Bromphenol blueBromphenol blue

--PyroninPyronin--PyroninPyronin

•Boja ne utiče na uzorak,•Boja ne utiče na uzorak,

putuje zajedno sa njim•putuje zajedno sa njim•putuje zajedno sa njim

•omogućava praćenje •omogućava praćenje

toka elektroforezetoka elektroforeze

TransferTransferTransferTransferTransferTransferTransferTransferaccessible to antibody detectionaccessible to antibody detection- In order to make the proteins accessible to antibody detectionaccessible to antibody detection:- In order to make the proteins accessible to antibody detectionaccessible to antibody detection:

they are moved from within the gel onto a membrane made of they are moved from within the gel onto a membrane made of they are moved from within the gel onto a membrane made of nitrocellulosenitrocellulose , nitrocellulosenitrocellulose ,

ppolyolyvviniledeninileden ddiiffluorideluoride (PVDF(PVDF)) orppolyolyvviniledeninileden ddiiffluorideluoride (PVDF(PVDF)) orppolyolyvviniledeninileden ddiiffluorideluoride (PVDF(PVDF)) or

nylon.nylon.nylon.nylon.nylon.nylon.

The membranemembrane is placed on top of the geltop of the gel, and a stack of filter stack of filter The membranemembrane is placed on top of the geltop of the gel, and a stack of filter stack of filter paperspapers placed on top of thattop of that.

The membranemembrane is placed on top of the geltop of the gel, and a stack of filter stack of filter paperspapers placed on top of thattop of that.paperspapers placed on top of thattop of that.

- The entire stack is placed in a buffer solution which moves up the paper by - The entire stack is placed in a buffer solution which moves up the paper by

capillary actioncapillary action, bringing the proteins with itproteins with it.capillary actioncapillary action, bringing the proteins with itproteins with it.capillary actioncapillary action, bringing the proteins with itproteins with it.

- Another method for transferring the proteins is called electrobloting and - Another method for transferring the proteins is called electrobloting and uses an electric currentelectric currentuses an electric currentelectric current to pull proteins from the gel into membrane.uses an electric currentelectric current to pull proteins from the gel into membrane.

All varieties of membrane are chosen for their All varieties of membrane are chosen for their All varieties of membrane are chosen for their

nonnon--specific protein binding propertiesspecific protein binding propertiesnonnon--specific protein binding propertiesspecific protein binding properties

(i.e. binds allall proteins equally well). (i.e. binds allall proteins equally well). (i.e. binds allall proteins equally well).

Protein binding is based upon hydrophobic interactionshydrophobic interactions,,Protein binding is based upon hydrophobic interactionshydrophobic interactions,,Protein binding is based upon hydrophobic interactionshydrophobic interactions,,

as well as charged interactionscharged interactions between the as well as charged interactionscharged interactions between the as well as charged interactionscharged interactions between the

membrane and protein.membrane and protein.membrane and protein.

- Nitrocellulose- Nitrocellulose membranes are cheapercheaper than- Nitrocellulose membranes are cheapercheaper than- Nitrocellulose membranes are cheapercheaper than

PVDF, but are far more fragilefar more fragile and PVDF, but are far more fragilefar more fragile and PVDF, but are far more fragilefar more fragile and PVDF, but are far more fragilefar more fragile and

do not stand up well to repeated do not stand up well to repeated probingsprobings.do not stand up well to repeated do not stand up well to repeated probingsprobings.do not stand up well to repeated do not stand up well to repeated probingsprobings.

One major difference between One major difference between One major difference between

nitrocellulose and PVDF membranes relates to the nitrocellulose and PVDF membranes relates to the nitrocellulose and PVDF membranes relates to the

ability of each to support ability of each to support "stripping" antibodies off"stripping" antibodies off and ability of each to support ability of each to support "stripping" antibodies off"stripping" antibodies off and ability of each to support ability of each to support "stripping" antibodies off"stripping" antibodies off and

reusingreusing the membrane for subsequent antibody probes.the membrane for subsequent antibody probes.reusingreusing the membrane for subsequent antibody probes.the membrane for subsequent antibody probes.

PVDF is sturdier, and can be more reused.PVDF is sturdier, and can be more reused.PVDF is sturdier, and can be more reused.PVDF is sturdier, and can be more reused.PVDF is sturdier, and can be more reused.PVDF is sturdier, and can be more reused.

Another difference is that, unlike nitrocellulose, PVDFAnother difference is that, unlike nitrocellulose, PVDFAnother difference is that, unlike nitrocellulose, PVDF

mustmust be soaked in 95% ethanol95% ethanol, isopropanolisopropanol ormustmust be soaked in 95% ethanol95% ethanol, isopropanolisopropanol ormustmust be soaked in 95% ethanol95% ethanol, isopropanolisopropanol or

methanol methanol before use. methanol methanol before use. methanol methanol before use.

PVDF membranes also tend to be thickerthicker and more more PVDF membranes also tend to be thickerthicker and more more PVDF membranes also tend to be thickerthicker and more more

resistant to damage during useresistant to damage during use.resistant to damage during useresistant to damage during use.resistant to damage during useresistant to damage during use.

The uniformity and overall effectiveness of transfer The uniformity and overall effectiveness of transfer The uniformity and overall effectiveness of transfer

of protein from the gel to the membrane can be of protein from the gel to the membrane can be of protein from the gel to the membrane can be

checked by staining the staining the membranemembrane with ponponcceau Seau S orchecked by staining the staining the membranemembrane with ponponcceau Seau S orchecked by staining the staining the membranemembrane with ponponcceau Seau S or

staining staining the the gelgel with coomassiecoomassie blueblue or SILvEr.SILvEr.staining staining the the gelgel with coomassiecoomassie blueblue or SILvEr.SILvEr.staining staining the the gelgel with coomassiecoomassie blueblue or SILvEr.SILvEr.

Ponceau S - češćačešća jer je osetljivija, osetljivija, i rastvorljiva u rastvorljiva u Ponceau S - češćačešća jer je osetljivija, osetljivija, i rastvorljiva u rastvorljiva u Ponceau S - češćačešća jer je osetljivija, osetljivija, i rastvorljiva u rastvorljiva u

vodivodi pa je kasnije lakše obezbojiti membranu vodivodi pa je kasnije lakše obezbojiti membranu vodivodi pa je kasnije lakše obezbojiti membranu

(reverzibilno bojenje nitroceluloze).(reverzibilno bojenje nitroceluloze).(reverzibilno bojenje nitroceluloze).

Coomassie blue se koristi samo za bojenje gela tj za bojenje proteina u Coomassie blue se koristi samo za bojenje gela tj za bojenje proteina u

gelu (snažno se vezuje za proteine, a ne za AA). Bojenje membrane gelu (snažno se vezuje za proteine, a ne za AA). Bojenje membrane gelu (snažno se vezuje za proteine, a ne za AA). Bojenje membrane

nije reverzibilno. Koristi za sagledavanje efikasnosti transfera nije reverzibilno. Koristi za sagledavanje efikasnosti transfera

proteina ili za sagledavanje separacije u gelu ako ne postoji namera proteina ili za sagledavanje separacije u gelu ako ne postoji namera proteina ili za sagledavanje separacije u gelu ako ne postoji namera

za transfer na membranu.za transfer na membranu.

Six general protein stains: Ponceau S colloidal goldSix general protein stains: Ponceau S colloidal gold

Coomassie blue colloidal silverCoomassie blue colloidal silver

amido black India inkamido black India ink

Blotting used to Blotting used to Blotting used to Analyzed Analyzed transfer the Analyzed Analyzed transfer the Analyzed Analyzed transfer the

samples from the through through samples from the through through samples from the through through

probing probing gel on to a probing probing gel on to a probing probing gel on to a with with membrane such with with membrane such with with

nucleic nucleic acid acid membrane such

as a nylon nucleic nucleic acid acid as a nylon nucleic nucleic acid acid as a nylon probesprobes or or membrane or probesprobes or or membrane or probesprobes or or

antibody antibody membrane or

nitrocellulose antibody antibody nitrocellulose antibody antibody nitrocellulose probesprobes..membrane. probesprobes..membrane. probesprobes..membrane.

*Blokada preostalih slobodnih mesta na membrani sa inertniminertnim proteinima.*Blokada preostalih slobodnih mesta na membrani sa inertniminertnim proteinima.*Blokada preostalih slobodnih mesta na membrani sa inertniminertnim proteinima.

steps must be taken to prevent interactions between the steps must be taken to prevent interactions between the steps must be taken to prevent interactions between the membranemembrane and the antibodyantibody used for detection of the target membranemembrane and the antibodyantibody used for detection of the target protein (since the antibody is a protein itself)(since the antibody is a protein itself).protein (since the antibody is a protein itself)(since the antibody is a protein itself).protein (since the antibody is a protein itself)(since the antibody is a protein itself).

Blocking of non-specific binding is achieved Blocking of non-specific binding is achieved

by placing the membrane in a dilute solution of proteinby placing the membrane in a dilute solution of protein

typically BBovinovinee SSerum erum AAlbumin lbumin (BSA)(BSA) or nonnon--fat dry milkfat dry milktypically BBovinovinee SSerum erum AAlbumin lbumin (BSA)(BSA) or nonnon--fat dry milkfat dry milktypically BBovinovinee SSerum erum AAlbumin lbumin (BSA)(BSA) or nonnon--fat dry milkfat dry milk

(both are inexpensive)(both are inexpensive), (both are inexpensive)(both are inexpensive), (both are inexpensive)(both are inexpensive),

with a minuteminute percentage of detergent such as Tween20Tween20.with a minuteminute percentage of detergent such as Tween20Tween20.

** This reduces ** The protein in the ** This reduces ** The protein in the ** This reduces ** The protein in the

dilute solution

** This reduces

"noise" in the dilute solution "noise" in the dilute solution

attaches to the attaches to the

"noise" in the

final product of attaches to the attaches to the final product of attaches to the attaches to the final product of attaches to the attaches to the

membrane in all membrane in all the Western blot, membrane in all membrane in all the Western blot, membrane in all membrane in all

placesplaces where the

the Western blot,

leading to clearer clearer placesplaces where the leading to clearer clearer placesplaces where the leading to clearer clearer

resultsresults, and placesplaces where the

target proteins have resultsresults, and target proteins have resultsresults, and target proteins have

not attached.

resultsresults, and

eliminates false eliminates false not attached. eliminates false eliminates false not attached. eliminates false eliminates false

positivespositives.positivespositives.positivespositives.

DetectionDetection-- Two stepTwo step DetectionDetection-- Two stepTwo step DetectionDetection-- OneOne stepstep DetectionDetection-- OneOne stepstep

the membrane is "probed" for the protein of interest with a modified antibody the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzymewhich is linked to a reporter enzyme, which when exposed to an

the membrane is "probed" for the protein of interest with a modified antibody which is linked to a reporter enzymewhich is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetriccolourimetric reactionreaction and produces a colourcolour.which is linked to a reporter enzymewhich is linked to a reporter enzyme, which when exposed to an appropriate substrate drives a colourimetriccolourimetric reactionreaction and produces a colourcolour.appropriate substrate drives a colourimetriccolourimetric reactionreaction and produces a colourcolour.

-- Two stepTwo step: 1. 2.: 1. 2.-- Two stepTwo step: 1. 2.: 1. 2.-- Two stepTwo step: 1. 2.: 1. 2.-Biotin iliDetectionDetection -Biotin ili

-Enzim (AP, HRP)+ supstrat

DetectionDetection

-Enzim (AP, HRP)+ supstrat

Primary antibodyPrimary antibody {Antibodies are generated when a host species or immune Primary antibodyPrimary antibody {Antibodies are generated when a host species or immune cell culture is exposed to the protein of interest (or a part thereof) }.cell culture is exposed to the protein of interest (or a part thereof) }.

A dilute solution of primary antibody (generally between 0.5 and 5 micrograms/(generally between 0.5 and 5 micrograms/mLmL)) is A dilute solution of primary antibody (generally between 0.5 and 5 micrograms/(generally between 0.5 and 5 micrograms/mLmL)) is incubated with the membraneincubated with the membrane under gentle agitation for anywhere from 30 minutes from 30 minutes incubated with the membraneincubated with the membrane under gentle agitation for anywhere from 30 minutes from 30 minutes to overnightto overnight at different temperaturesdifferent temperatures.to overnightto overnight at different temperaturesdifferent temperatures.

The solution is composed of buffered saline solutionbuffered saline solution with a small percentage of The solution is composed of buffered saline solutionbuffered saline solution with a small percentage of detergentdetergent, and sometimes with powdered milk or BSA. powdered milk or BSA. detergentdetergent, and sometimes with powdered milk or BSA. powdered milk or BSA.

Secondary antibodySecondary antibody {After rinsing the membranerinsing the membrane to remove Secondary antibodySecondary antibody {After rinsing the membranerinsing the membrane to remove

unbound primary antibody, the membrane is exposed to unbound primary antibody, the membrane is exposed to unbound primary antibody, the membrane is exposed to

another antibody, directed at a species-specific portion of the another antibody, directed at a species-specific portion of the another antibody, directed at a species-specific portion of the

primary antibody }.primary antibody }.

The secondary antibody is usually linked to biotinbiotin or to a reporter reporter The secondary antibody is usually linked to biotinbiotin or to a reporter reporter The secondary antibody is usually linked to biotinbiotin or to a reporter reporter

enzymsenzyms such as alkalinalkalin phosphatasephosphatase or horseradish horseradish peroxidaseperoxidase. enzymsenzyms such as alkalinalkalin phosphatasephosphatase or horseradish horseradish peroxidaseperoxidase. enzymsenzyms such as alkalinalkalin phosphatasephosphatase or horseradish horseradish peroxidaseperoxidase.

This means that several secondary antibodies will bind to one This means that several secondary antibodies will bind to one

primary antibody and enhance the signal.primary antibody and enhance the signal.primary antibody and enhance the signal.

Most commonly, a horseradish peroxidase-linked secondary is Most commonly, a horseradish peroxidase-linked secondary is Most commonly, a horseradish peroxidase-linked secondary is

used to cleave a cleave a chemiluminescentchemiluminescent agentagent, and the reaction used to cleave a cleave a chemiluminescentchemiluminescent agentagent, and the reaction

product produces luminescenceluminescence in proportion to the amount of amount of product produces luminescenceluminescence in proportion to the amount of amount of product produces luminescenceluminescence in proportion to the amount of amount of

proteinprotein.proteinprotein.

A cheapercheaper but less sensitiveless sensitive approach utilizes a A cheapercheaper but less sensitiveless sensitive approach utilizes a A cheapercheaper but less sensitiveless sensitive approach utilizes a

44--chloronaphtholchloronaphthol stainstain with 1% horseradish 1% horseradish peroxidaseperoxidase; 44--chloronaphtholchloronaphthol stainstain with 1% horseradish 1% horseradish peroxidaseperoxidase;

reaction of peroxide radicals with 4-chloronaphthol produces reaction of peroxide radicals with 4-chloronaphthol produces reaction of peroxide radicals with 4-chloronaphthol produces

a dark brown staina dark brown stain that can be photographed a dark brown staina dark brown stain that can be photographed

without using specialized photographic film.without using specialized photographic film.without using specialized photographic film.

-- One stepOne step-- One stepOne step-- One stepOne stepHistorically, the probing process was performed in two steps Historically, the probing process was performed in two steps

because of the relative ease of producing primary and Historically, the probing process was performed in two steps

because of the relative ease of producing primary and because of the relative ease of producing primary and secondary antibodies in separate processes.secondary antibodies in separate processes.

oneone--step probing systems that would allow the process step probing systems that would allow the process oneone--step probing systems that would allow the process step probing systems that would allow the process oneone--step probing systems that would allow the process step probing systems that would allow the process to occur to occur fasterfaster and with and with less consumablesless consumables.to occur to occur fasterfaster and with and with less consumablesless consumables.to occur to occur fasterfaster and with and with less consumablesless consumables.

probe antibodyprobe antibodyThis requires a probe antibodyprobe antibody which both recognizes the This requires a probe antibodyprobe antibody which both recognizes the This requires a probe antibodyprobe antibody which both recognizes the protein of interest and contains a detectable label, probes protein of interest and contains a detectable label, probes which are often available for known proteins tags.proteins tags.which are often available for known proteins tags.proteins tags.which are often available for known proteins tags.proteins tags.

Analysis :Analysis :Analysis :Analysis :Analysis :Analysis :Analysis :Analysis :In practical terms, not all Westerns reveal protein only at In practical terms, not all Westerns reveal protein only at

one bandone band in a membrane.In practical terms, not all Westerns reveal protein only at

one bandone band in a membrane.one bandone band in a membrane.

Size approximations are taken by comparing the stained comparing the stained Size approximations are taken by comparing the stained comparing the stained bandsbands to that of the marker or ladder loadedmarker or ladder loaded during

Size approximations are taken by comparing the stained comparing the stained bandsbands to that of the marker or ladder loadedmarker or ladder loaded during bandsbands to that of the marker or ladder loadedmarker or ladder loaded during electrophoresiselectrophoresis.electrophoresiselectrophoresis.electrophoresiselectrophoresis.

The process is repeated for a structural proteinstructural protein, such as The process is repeated for a structural proteinstructural protein, such as actin or tubulin, that should not change between

The process is repeated for a structural proteinstructural protein, such as actin or tubulin, that should not change between actin or tubulin, that should not change between samples.samples.samples.

This practice ensures correction for the amount of total ensures correction for the amount of total This practice ensures correction for the amount of total ensures correction for the amount of total This practice ensures correction for the amount of total ensures correction for the amount of total proteinprotein on the membrane in case of errors or proteinprotein on the membrane in case of errors or incomplete transfers.proteinprotein on the membrane in case of errors or incomplete transfers.incomplete transfers.

Colorimetric detectionColorimetric detection ::Colorimetric detectionColorimetric detection ::Colorimetric detectionColorimetric detection ::

This method depends on This method depends on BCIP/NBT Alkaline Phosphatase Substrate SolutionThis method depends on

incubation of the Western blot with a incubation of the Western blot with a substratesubstrate

BCIP/NBT Alkaline Phosphatase Substrate Solution

incubation of the Western blot with a incubation of the Western blot with a substratesubstrateincubation of the Western blot with a incubation of the Western blot with a substratesubstrate

that reacts with the reporter that reacts with the reporter enzymeenzyme (such as (such as peroxidaseperoxidase or APor AP))that reacts with the reporter that reacts with the reporter enzymeenzyme (such as (such as peroxidaseperoxidase or APor AP))that reacts with the reporter that reacts with the reporter enzymeenzyme (such as (such as peroxidaseperoxidase or APor AP))

that is bound to the secondary antibody.secondary antibody.that is bound to the secondary antibody.secondary antibody.that is bound to the secondary antibody.secondary antibody.

This converts the solublesoluble dye This converts the solublesoluble dye

into an insolubleinsoluble form of a into an insolubleinsoluble form of a

different color that PRECIPITATESPRECIPITATES next to the enzyme different color that PRECIPITATESPRECIPITATES next to the enzyme

and thereby stains the membrane.and thereby stains the membrane.

Protein levels are evaluated through Protein levels are evaluated through Protein levels are evaluated through

densitometrydensitometry spectrophotometryspectrophotometrydensitometrydensitometry or spectrophotometryspectrophotometry.densitometrydensitometry or spectrophotometryspectrophotometry.

ChemiluminescentChemiluminescent detection :detection :Chemiluminescent substrates for

ChemiluminescentChemiluminescent detection :detection :Chemiluminescent substrates for

horseradish peroxidase (HRP) ChemiluminescentChemiluminescent detection :detection : horseradish peroxidase (HRP)

are two-component systems ChemiluminescentChemiluminescent detection :detection :

are two-component systems

consisting of consisting of

1. a stable peroxide solution and

This methods depend on

1. a stable peroxide solution and

2. an enhanced luminol solution.This methods depend on 2. an enhanced luminol solution.This methods depend on

incubation of the Western blot with a incubation of the Western blot with a substratesubstrateincubation of the Western blot with a incubation of the Western blot with a substratesubstrateincubation of the Western blot with a incubation of the Western blot with a substratesubstrate

that that will will luminesceluminesce when exposed when exposed to the reporter on the to the reporter on the 22°° AbAb..that that will will luminesceluminesce when exposed when exposed to the reporter on the to the reporter on the 22°° AbAb..that that will will luminesceluminesce when exposed when exposed to the reporter on the to the reporter on the 22°° AbAb..

The light is then detected by photographic filmphotographic film or CCDCCD camerascameras. The light is then detected by photographic filmphotographic film or CCDCCD camerascameras. The light is then detected by photographic filmphotographic film or CCDCCD camerascameras.

The image is The image is analysedanalysed by densitometry.by densitometry.The image is The image is analysedanalysed by densitometry.by densitometry.The image is The image is analysedanalysed by densitometry.by densitometry.

Newer software allows further data analysis such as molecular molecular Newer software allows further data analysis such as molecular molecular

weight analysisweight analysis if appropriate standards are used.weight analysisweight analysis if appropriate standards are used.weight analysisweight analysis if appropriate standards are used.

Radioactive detection :Radioactive detection :Radioactive detection :Radioactive detection :Radioactive detection :Radioactive detection :

Radioactive labels do not require enzyme substratesdo not require enzyme substrates, Radioactive labels do not require enzyme substratesdo not require enzyme substrates, Radioactive labels do not require enzyme substratesdo not require enzyme substrates,

but rather allow the placement of medical Xmedical X--ray film ray film but rather allow the placement of medical Xmedical X--ray film ray film but rather allow the placement of medical Xmedical X--ray film ray film

directly against the western blotdirectly against the western blot which develops as it directly against the western blotdirectly against the western blot which develops as it directly against the western blotdirectly against the western blot which develops as it

is exposed to the label and creates dark regionsdark regionsis exposed to the label and creates dark regionsdark regionsis exposed to the label and creates dark regionsdark regions

which correspond to the protein bands of interestcorrespond to the protein bands of interestwhich correspond to the protein bands of interestcorrespond to the protein bands of interestwhich correspond to the protein bands of interestcorrespond to the protein bands of interest

very expensive, health and safety risks are highvery expensive, health and safety risks are highvery expensive, health and safety risks are high

Fluorescent detection :Fluorescent detection :Fluorescent detection :Fluorescent detection :Fluorescent detection :Fluorescent detection :Fluorescent detection :Fluorescent detection :

The fluorescently labeled probe is excited by lightlightThe fluorescently labeled probe is excited by lightlightThe fluorescently labeled probe is excited by lightlightand the emission of the excitation is then the emission of the excitation is then and the emission of the excitation is then the emission of the excitation is then and the emission of the excitation is then the emission of the excitation is then detected by a detected by a photosensorphotosensor such as CCD camerasuch as CCD camera. detected by a detected by a photosensorphotosensor such as CCD camerasuch as CCD camera. detected by a detected by a photosensorphotosensor such as CCD camerasuch as CCD camera.

Allows further data analysis such as molecular molecular Allows further data analysis such as molecular molecular Allows further data analysis such as molecular molecular weightweight analysis and a quantitative western blot quantitative western blot weightweight analysis and a quantitative western blot quantitative western blot weightweight analysis and a quantitative western blot quantitative western blot analysisanalysis. analysisanalysis. analysisanalysis.

the most sensitivemost sensitive detection methods for blotting the most sensitivemost sensitive detection methods for blotting the most sensitivemost sensitive detection methods for blotting analysis.analysis.analysis.

Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:

• Detekcija At• Detekcija At• Detekcija At

Inkubacija traka sa ispitivanim serumom, a Inkubacija traka sa ispitivanim serumom, a Inkubacija traka sa ispitivanim serumom, a

potom sa anti-At koja su konjugovana sa enzimom.potom sa anti-At koja su konjugovana sa enzimom.potom sa anti-At koja su konjugovana sa enzimom.

Na kraju se dodaje supstratNa kraju se dodaje supstrat

• Detekcija Ag• Detekcija Ag• Detekcija Ag

U smeši proteina dobijenih liziranjem μ.o. (k.ć. saU smeši proteina dobijenih liziranjem μ.o. (k.ć. saU smeši proteina dobijenih liziranjem μ.o. (k.ć. sa

virusom, bakterije?), identifikuju se Ag upotrebom At virusom, bakterije?), identifikuju se Ag upotrebom At virusom, bakterije?), identifikuju se Ag upotrebom At

obeleženih enzimima.obeleženih enzimima.obeleženih enzimima.

Npr. Dg HIVHIV infekcije analizom seruma na At:Npr. Dg HIVHIV infekcije analizom seruma na At:Npr. Dg HIVHIV infekcije analizom seruma na At:

Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:Medical diagnostic applications:

- definitive test for - definitive test for - definitive test for

Bovine Spongiform Encephalopathy (BSE).Bovine Spongiform Encephalopathy (BSE).Bovine Spongiform Encephalopathy (BSE).Bovine Spongiform Encephalopathy (BSE).Bovine Spongiform Encephalopathy (BSE).Bovine Spongiform Encephalopathy (BSE).

-Some forms of Lyme disease testing employ Western blotting.-Some forms of Lyme disease testing employ Western blotting.-Some forms of Lyme disease testing employ Western blotting.

((The confirmatory HIV testThe confirmatory HIV test))((The confirmatory HIV testThe confirmatory HIV test))((The confirmatory HIV testThe confirmatory HIV test))

-Western blot can also be used as a confirmatory test for Hepatitis B infection. -Western blot can also be used as a confirmatory test for Hepatitis B infection. -Western blot can also be used as a confirmatory test for Hepatitis B infection.

AdvantagesAdvantagesAdvantagesAdvantagesAdvantagesAdvantages

Specific interaction of antibody and antigen can Specific interaction of antibody and antigen can Specific interaction of antibody and antigen can

be directly visualized.be directly visualized.be directly visualized.

DisadvantagesDisadvantagesDisadvantages

Technically demandingTechnically demandingTechnically demanding

ExpensiveExpensiveExpensive

Subject to interpretationSubject to interpretationSubject to interpretation

Presence or absence of bandsPresence or absence of bandsPresence or absence of bands

Intensity of those bandsIntensity of those bandsIntensity of those bands

When should WB be used?When should WB be used?When should WB be used?When should WB be used?When should WB be used?When should WB be used?

screening testscreening testWB assay should not be used as a - screening testscreening test.WB assay should not be used as a - screening testscreening test.

WB should be viewed as a supplemental testsupplemental test which can be WB should be viewed as a supplemental testsupplemental test which can be WB should be viewed as a supplemental testsupplemental test which can be

used to confirm positive results obtained from confirm positive results obtained from EIAEIA**used to confirm positive results obtained from confirm positive results obtained from EIAEIA***Enzyme immunoassay*Enzyme immunoassay

HOWEVER:HOWEVER:

!!!!!!SpecificitySpecificity is less thanless than that of EIA !!!!!!SpecificitySpecificity is less thanless than that of EIA !!!!!!A significant number of indeterminate blots are seen in low risk A significant number of indeterminate blots are seen in low risk A significant number of indeterminate blots are seen in low risk

populationspopulations