Molecular Pathology LAB

PRNT, SDS-PAGE,
WESTERN BLOT
By: Rida Eka & Zidni Hasbuna

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Plaque reduction neutralizing test (PRNT) :

is currently the ‘gold standard’ for the
differential serodiagnosis of flaviviruses and
to quantify the titer of neutralizing antibodies
against flaviviruses.

Objective :
Determine the flavivirus neutralizing antibodies in
serum sample.
 CELL CULTURE

Discard media Wash the cell with 1X PBS buffer Add 0.25% trypsin EDTA

Make a single
cell with
pippeting

Incubate at 37°C, 5%
Examine cell under microscope CO2 for 5 minute

1. Seed LLC-MK2 onto 6-well plate for DENV 1-4

2. Seed Vero cells onto 6-well for ZIK-V and JEV
Inactivate trypsin using completed 3. Incubate cell at 37°C, 5% CO2 overnight
media containing FBS
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 Cell morphology

Vero cell LLC-MK2 cell

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 PRNT 6

Blood collection Incubate serum diluted serum diluted virus

Incubate serum+virus
at 37oC for 1 hrs

Incubate cell with Add diluted virus and

mixture at 37C for 2 h serum mixture onto cells

37C , 5% CO2
for 15-17 h

Overlay cells with 1X

Incubate cells at 37oC Add secondary overlay solution Count plaque number
nutrient containing 0.8% or
with 5% CO2 for 6 days containing 0.5% neutral red
0.3% agarose and interprete PRNT
COUNT RESULT :

RIDA ZIDNI
virus PRNT50 PRNT90 PRNT50 PRNT90

JEV
80 < 20 80 40
CONCLUSION :
ZIKV - - < 20 < 20

1. For Rida antibody can

DENV-1 > 320 > 320 < 20 < 20 neutralizing 5 virus

2. For Zidni antibody can

DENV-2 > 320 > 320 < 20 < 20
neutralizing only JEV

DENV-3 > 320 40 < 20 < 20

DENV-4 > 320 40 < 20 < 20

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SDS-PAGE Sodium Dodecyl Sulfate –
Polyacrylamide Gel Electrophoresis
Variant of polyacrylamide gel electrophoresis
to separation of charged molecules in
mixtures by their molecular masses in
an electric field. It uses sodium dodecyl
sulfate (SDS) molecules to help identify and
isolate protein molecules
SAMPLE PREPARATION

PROTEIN SAMPLE
DENATURE BUFFER / -Control
-EGFP-C2 (Green Flourescent Protein)
LOADING DYE -Full Length GRP 78 (Containing of GFP, NBD, SBD)
-NBD (Nucleotide binding domain)
-SBD (Substrated binding domain)

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 - Stacking Gel 5%

Separating Gel 12%

All Blue Protein Sample
Protein
Marker
+
Acrylamide Gel
Polyacrylamide Gel

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 Wait until loading dye migrate
120 volts to the end of gel

Coomassie blue

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Only separating Cut the stacking gel

gel

Incubate
Overnight

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 CONCLUSION:
Control EGFP FULL NBD SBD
LENGTH
The total protein is show
250 kD in the SDS PAGE (in a band)
150
100 by staining with the coomesie
75 blue dye
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37

25

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WESTERN BLOT
 A technique to
detect a specific
protein in a protein
mixture
 Applay the electrical field

-----------------
Prepare the protein sample Put the solution in
and loading dye the pore
Cut the stacking gel

Only separating gel

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 ------------
The membrane
with the protein

Add 5% skim milk

Primary
Antibody

Rabbit anti-GFP Rabbit anti-GRP 78

antibodies antibodies

Incubate membrane in Transfer the protein onto the membrane

overnight at Cold Room with tank blotting system
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 3X

Chemiluminescence
Wash it with TBS
Secondary
Antibody

Luminor
Enhancer
Solution
Goat Anti Rabbit IgG-HRP

3X

Wash again

Incubate membrane for 2 h

TBS 18
 GRP 78 GRP 78
FULL FULL
GFP LENGTH NBD SBD Cntl EGFP LENGTH NBD SBD

250 kD
150
100

75

50

37

25

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Rabbit anti-GFP antibodies Rabbit anti-GRP 78 antibodies

( 1 : 1000 ) GFP : Green Flourescent Protein ( 1 : 1000 )
NBD : Nucleotide binding domain
SBD : Substrated binding domain
FULL LENGTH : Containing of GFP, NBD, SBD 19
 Thanks to :
Prof. Duncan R.Smith
Dr. Wannapa Sornjai
Janejira Jaratsittisin
All Staff in Molecular Pathology LAB

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