Transcript 2004 Dot blotting presentation by Chng-Tau, Poppy, and

Fundamental Of Bioprocess Engineering 1
Dot Blotting
Ch-ng Tau
Poppy
Yang
What Is Dot Blotting
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It is introduced in 1970s, to identify antigens
that bound to specific antibodies
It can be used either as a qualitative method
for rapid screening of large number of samples
or as a quantitative technique
Many different way to do dot blotting, e.g
Electroblotting
Many detection methods e.g.Radioactive
We are using the simplest and cheapest way
--manual spotting qualitative method
Theoretical Background
Aim of the experiment: identify ovalbumin among
casein and gluten
Objective of the experiment : produce a stain on
the nitrocellulose membrane
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Proteins are complicated amino acid sequence,
have high molecular weights, classified
according to biological function, source and
occurrence
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Antibody are amino acid sequence too, can
attach to the specific protein
Theoretical Background
Add PA
P1
P2
P3
P1
P2
P3
PA
PA
PA
Add SA
Add
developing
solution
P1
SPA
P2
P3
SPA
SPA
Theoretical Background
Developing solution is added to produce
stain: in the developing solution, in the
presence of hydrogen peroxide, dye is
oxidised and form an insoluble white
coloured compound on the ovalbumin
dot
 As secondary antibody occupy the whole
membrane, developing solution make
the membrane to blue colour.
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In This Experiment
Proteins used:casein, ovalbumin, and
gluten----10ml, 0.1 %(w/v) in urea
 Blocking solution: BSA (Bovine Serum
albumin)---250ml, 2 %(w/v) in Tris-Cl
 Primary antibody:anti chicken egg
(ovalbumin) developed in rabbit---12ml,
0.01%(w/v) in Tris-Cl
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In This Experiment
Secondary antibody:anti rabbit IgG
developed in goat---12ml, 0.01 %(w/v) in
Tris-Cl
 Developing solution:100ml, contains
0.48mM 4-Chloro –naphthol, 17%(v/v)
methanol, and need to add H2O2 at
concentration of 0.01%(v/v) just before
we need to use developing solution
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Procedure
Cut 1×8cm nitrocellulose paper strips
 Prepare solutions needed in the
experiment
 Concentration of antibody solutions can
be much smaller than protein solutions,
as the reaction is very sensitive, and we
can save some of the expensive
antibodies
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Procedure
Eject 2µL each for the three different protein
solutions along the strips IN AN ORDER!
Or you may be very confused when facing the
result!
Be very careful to avoid contamination of protein
sample!
C
O
G
Procedure
Soak in blocking
solution
Dry the dots
10min
Soak in
Tris-Cl
5min
Soak in
secondary
antibody
5min
Soak in
Tris-Cl
1hour
Soak in
primary
antibody
1hour
Soak in
Tris-Cl
5min
What we need to do in this 5min
waiting time?….see next slide
please!
Procedure
Prepare the developing solution quickly
and place the strip into it immediately
 Look it up after 10min to find out if there
is any colour developed
 The expect result is:
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C
O
G
How Did We Do The Experiment
We actually did not prepare the solutions
we need in exact concentration
 We did not soak the strip in the solutions
as long as it suppose to be as shown in
the procedure
 During the experiment, we make a lot
mistakes( say scratch the strip), which
may affect the result
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Experiment Result
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We did not get the expected result!
C
O
G
the gluten’s dot show blue—good!
 However, the casein dot suppose to be
blue as well—negative result!
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Discussion
Casein may not blocked to the strip, or
may be blocked but got scratched off?
 Antibody did not occupy the casein dot?
 Perhaps when the strips were being
dried, casein deposited out as the solid,
and show the colour in the later steps?
 casein was contaminated by ovalbumin?
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Further Discussion
Be more accurate and careful when
doing the experiment.
 Leave the strip in all the solutions longer.
 Do further research to improve the
experiment (say use a different way or
solution?)
 Quantitative test to the strip can be
applied, as a extension to the qualitative
method that we have so far
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