Transcript Where do experimental antibodies come from?

The University
of Manchester
BIOL20332/20972
GENETICS / Dev. Biol.
RSM
Faculty of
Life Sciences
MODULE 2
Immuno
histochemistry
Andreas Prokop
Drosophila developmental stages
EXPERIMENTAL OBJECTIVE of week 2
To understand how molecular mechanisms of axonal guidance can be
studied via genetic loss-of-function analysis combined with
immunohistochemistry. For this, you will study nervous system
defects of selected Drosophila mutant embryos.
• What do experimental
antibodies detect?
• Where do experimental
antibodies come from?
• How to perform the
experiment!
Differential gene expression - regulating genes at different levels
Gene
transcription (epigenetics,
TFs, differential start sites)
UTR EX IN
primary
transcript
non-coding
primiRNAs
RNA processing
(spliceosome, alternative splicing)
mRNA
miRNAs
translation (ribosomes, initiation,
elongation, termination factors)
protein (2nd)
folding, complex formation
(chaperones)
protein
(tertiary, quaternary)
posttranslational
modifications (enzymes)
protein***
(phosphorylation, glycosylation, ubiquitination...)
protein***
• What do experimental
antibodies detect?
• Where do experimental
antibodies come from?
• How to perform the
experiment!
Immune response
Antibodies
http://www.news-medical.net/health/Antibody-Function.aspx
How to obtain antibodies
polyclonal antibodies
monoclonal antibodies
injecting
antigen X
injecting antigen X
1st boost
2nd boost
bleed +
purification
cells producing
anti-X antibody
producing
hybridoma
cells
Use of secondary antibodies
rabbit
antibody
donkey
anti-rabbit
mouse
antibody
donkey
anti-mouse
Making antibodies visible
crisp, excellent
crisp +
double-labelling,
permanent,
but not
double-labelling
permanent
less optimal
incremental but
diffuse; mostly in
situ hybridisation
only EM, usually
loss of tissue
contrast (due to
detergent)
ABC enhancement kit
H2N
NH2
H2N
diaminobenzidine (DAB)
NH2
• What do experimental
antibodies detect?
• Where do experimental
antibodies come from?
• How to perform the
experiment!
Summary of the procedure
- fixation (PF)
- detergent (PBT)
- 1st antibody
- wash (PBT)
- 2nd antibody
- wash (PBT)
- ABC kit
- wash (PBT)
- DAB/H202
Remove chorion:
Remove chorion:
Images: Christoph Rickert
Transfer into the sieve:
pour the bleach
with the eggs into
the sieve and wash
with water
Images: Christoph Rickert
Transfer into fix:
transfer eggs with a brush
Images: Christoph Rickert
Prepare fixative:
• Everybody prepares a microfuge tube with fixation solution
BEFORE STARTING THE WHOLE PROCEDURE
• label the tube appropriately (group, genotype, antibody)
500µl 4%
formaldehyde
in PBS
Images: Christoph Rickert
Incubate in fix for 30 min:
Images: Christoph Rickert
Fixation
Schiff base: ketimine,
condensation product of
a carbonyl group of an
aldehyde or keton with
an amino group
Apart from cross-linking agent, proteins can be
denatured/fixed through different means:
• Acids (e.g. acetic acid)
• solvents (e.g. ethanol, methanol)
• heat (e.g. 1 min 60°C)
Remove PBS/fix (lower phase):
Images: Christoph Rickert
Add methanol:
Add 700µl of
methanol
Shake vigorously
for 20 seconds!!
Images: Christoph Rickert
After embryos sunk to the bottom:
• Remove all liquid (but not embryos!!!) and fill up
with methanol
• Remove methanol and wash again with fresh
methanol
• Exchange methanol for PBT
• Wash once more with PBT
• Add primary antibody
Distribution of fly stocks across the course
mutant
gene
BP102
(mouse)
A
groups
XXX
groups
XXX
B
groups
XXX
groups
XXX
C
groups
XXX
groups
XXX
C-lacZ
anti-A
(mouse)
anti-C
(mouse)
anti-βGal
(rabbit)
groups groups groups
XXX
XXX
XXX
anti-Fas2
(mouse)
Source of egg lays
Each group gets different batches of egg lays of the same genotype:
batch labelled No 1
Sat 6pm - Sun 9am  stored 12ºC
batch labelled No 2
Sun 9am - 2pm  stored 18ºC
batch labelled No 3
Sun 2 - 7pm  stored 20ºC, 12ºC since Mon 10am
batch labelled No 4
Sun 7pm - Mon 10am  stored 25ºC, 12ºC since Mon 10am
Short guide to the laboratory protocol
Consider that in a real laboratory situation you will not have the time to
write long texts and explanations. Therefore, find economic ways that
are nevertheless understandable - not only to you, but also to others.
a) PAGE NUMBER and DATE
b) AIM/RATIONALE OF EXPERIMENT
c) Details about your experimental objects. (GENOTYPE, AGE or
DEVELOPMENTAL STAGE)
d) Details about MATERIALS/CHEMICALS (e.g. fixatives, antibodies
etc.).
e) SINGLE STEPS OF YOUR EXPERIMENTS (note that clear
reference to the manual may suffice to cover methods)
f) SPECIAL OBSERVATIONS, PROBLEMS, TIPS, TRICKS,
EXPLANATIONS or THOUGHTS
g) REFERENCES TO EXTERNAL SOURCES (pages in manual,
location of specimens, existence of documentation)
h) OUTCOME at intermediate stages and upon termination of the
experiment