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Which plant is transgenic?

Determination using an
enzyme-linked immunosorbent assay
transgenic plants / GMOs
A transgenic plant has incorporated gene(s)
from another species.
Transgenics are useful for engineering a
specific phenotype or studying a genes
function.


transgenic selection: NPTII
Selection of plants that incorporated the transgene
can be performed by simultaneously introducing a
drug resistance gene into the plant (just like we do
with E. coli).
nptII (most common) codes for the enzyme
aminoglycoside 3'-phosphotransferase (NPTII)
that phosphorylates (inactivates) aminoglycoside
antibiotics including kanamycin, neomycin
geneticin (G418) and paromomycin.
transgenic selection:
kanamycin resistance due to NPTII
kanamycin
WT
kanamycin
Transgenic
NPTII
Which plant is transgenic?
Which plant is transgenic?
WT WT WT transgenic
Need both WT and transgenic plants for
experiment (today) so kanamycin selection is out.
Perform test for transgene or transgene protein?
ELISA is a fast way to test for the presence and
amount of a protein such as NPTII.
ELISA
understanding the acronym
EL Enzyme-linked
An enzymes activity is used as a reporter
to test for the presence/amount of a protein
of interest.
The enzymatic reaction will produce a
colored species.


ELISA
understanding the acronym
EL Enzyme-linked
IS Immunosorbent
An antibody or antigen (immuno) is
adsorbed (sorbent) onto the polystyrene
wells in which we conduct the test.
One antibody is already adsorbed added to
the wells and a second antibody with our
enzyme will be added in the type of ELISA
we will perform today.

ELISA
understanding the acronym
EL Enzyme-linked
IS Immunosorbent
A Assay
We will could determine the amount of
NPTII (quantitative assay).
We will use NPTII concentration standards
to construct a standard curve.
ELISA: uses antibodies
What is an antibody?
What types of antibodies exist?
What kinds of antibodies are used in our lab
today?
What is an antibody?
Protein secreted by B-cells that
specifically bind a foreign
substance (antigen)
Immunoglobulin domains
Complementarity-determining
Regions (CDRs)
Fab= Fragment antigen binding
Hinge
Fc= Fragment crystalline
F(ab)2= Protease digestion still
useful to bind antigen

producing polyclonal antibodies
producing monoclonal antibodies
1
2
3
Antibody-based assays
Ag Ag
Types of immunodetection systems
1. Direct immunodetection
Primary antibody conjugated with
enzyme system
2. Indirect immunodetection
Secondary antibody conjugated with
enzyme system
3. Sandwich indirect
immunodetection
Antigen applied in soluble form
4. Indirect immunodetection
with biotin linkers
Biotinylated primary antibodies
Ag Ag
Ag Ag
HRP
HRP
HRP
HRP
HRP
HRP
HRP
HRP
Ag Ag
HRP
Streptavidin
HRP
HRP HRP
HRP
streptavidin
HRP
horseradish
peroxidase
antigen
Ag
Todays assay
Sandwich indirect
immunodetection

Antigen applied in soluble form

HRP
HRP
HRP
HRP
Substrate
Substrate
Substrate
Substrate
Sandwich ELISA protocol
1. Coat primary antibody
onto microplate.
1a. Allow antibody adsorption
and block unoccupied sites
with neutral protein (BSA).
2. Add antigen sample to be detected
into each well. Incubate 30 min at 37
0
C.
4. Develop colorimetric reaction
with appropriate substrate. Incubate
15 min at room temperature.
5. Stop reaction with 3M H
2
SO
4
. Read
absorbance in ELISA spectrophotometer
and quantitate relative antigen levels.
3. Add second primary antibody
against antigen and HRP-conjugated
secondary antibody (antibody mix)
into each well. Incubate 30 min at 37
0
C.
What you will do today (1):
Collect and weigh tissue sample plant A, B and C.
Repeat for second and third plants.
Add 400 L PEBX1 buffer to each microcentrifuge
tube and grind plant tissue using pestle.
Add 100 L of PEBX1 to wells A and B.
Add 100 L of one sample to wells C and D.
Add 100 L of second sample to wells E and F.
Add 100 L of third sample to wells G and H.
HANDLE THE WELLS LIKE A CUVETTE (read at
bottom)





A B C D E F G
H
What you will do today (2):
Incubate 30 minutes at 37 C.
Place wells in a zip-close bag to prevent them from
drying out in the oven.
Wash wells 3 times with PBST
Add antibody-enzyme conjugate (MRS-2 Ab)
Note the dilution factor for the conjugate
Incubate 30 minutes at 37 C.
Place wells in a zip-close bag to prevent them from
drying out in the oven.
Wash 4 times with PBST.
What you will do today (3):
Add substrate and allow blue color to develop.
Stop the enzymatic reaction with 3M H
2
SO
4
.

Presence of NPTII will result in color change from
blue to yellow.
How we will detect:
read absorbance at 450 nm
Data
Absorbance 450 nm
1 2 3 4 5 6 7 8 9 10 11 NPTII Std


W
E
L
L
A
B
C
D
E
F
G
H
Data analysis (due 4/23/12)
Total
amount of
plant tissue
(mg) A450
NPTII
concentration
(ng/mL)
NPTII amount
(ng protein
/mg tissue) Transgenic? A 450
NPTII
concentration
(ng/mL)
Your
Sample
Your
Sample Your Sample Your Sample Your sample Standard Standard


W
E
L
L
A Blank N/A 0 N/A N/A
2
B Blank N/A 0 N/A N/A
1
C Plant __
0.5
D Plant __ Average Average Yes/No
0.25
E Plant __
0.125
F Plant __ Average Average Yes/No
0.0625
G Plant __
0.03125
H Plant __ Average Average Yes/No
0.015625
y = 0.3631x - 0.0025 R = 0.995
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.5 1 1.5 2
a
b
s
o
r
b
a
n
c
e

a
t

4
5
0

n
m

NPTII standard (ng/mL)

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