Transcript Where do experimental antibodies come from?

Faculty of
Life Sciences
The University
of Manchester
BIOL22332/20972
Genetics / Dev.Biol
RSM
MODULE 2 (Andreas Prokop)
Immunohistochemistry
Drosophila developmental stages
Immunohistochemical analyses performed during this course
EXPERIMENTAL OBJECTIVE of week 2:
The aim of this experiment is to describe nervous system defects of selected
Drosophila mutant embryos, in order to illustrate molecular mechanisms that
regulate axonal CNS midline crossing - and the experimental use of genetic
loss-of-function analysis and immunohistochemistry.
• What do experimental
antibodies detect?
• Where do experimental
antibodies come from?
• How does immunocyto/histochemistry work?
Differential gene expression - equipping cells with the right set of gene products
non-coding
Gene
transcription (epigenetics,
TFs, differential start sites)
UTR EX IN
primary
transcript
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...)
degradation (proteasome, lysosome)
protein***
Detecting gene products at different levels
non-coding
Gene
transcription (epigenetics,
TFs, differential start sites)
in situ hybridisation
with intron probes
primary
transcript
primiRNAs
RNA processing
(spliceosome, alternative splicing)
in situ hybridisation
with exon probes
immunochemistry
mRNA
translation (ribosomes, initiation,
elongation, termination factors)
protein (2nd)
folding, complex formation
(chaperones)
immunochemistry
protein
(tertiary, quaternary)
posttranslational
modifications (enzymes)
immunochemistry
mass spectrometry
miRNAs
protein***
(phosphorylation, glycosylation, ubiquitination...)
degradation (proteasome, lysosome)
protein***
• What do experimental
antibodies detect?
• Where do experimental
antibodies come from?
• How does immunocyto/histochemistry work?
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
double-labelling,
but not
permanent
crisp +
permanent,
double-labelling
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 does immunocyto/histochemistry work?
Summary of procedure
- fixation (PF)
- detergent (PBT)
- 1st antibody
- wash (PBT)
- 2nd antibody
- wash (PBT)
- ABC kit
- wash (PBT)
- DAB/H202
Remove chorion:
Images: Christoph Rickert
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
mutant
gene
BP102
(mouse)
A
groups
B
groups
C
B
groups
DE
groups
I
C
groups
FG
groups
H
C-lacZ
anti-A
(mouse)
anti-C
(mouse)
anti-βGal
(rabbit)
groups groups groups
KL
MN
OP
anti-Fas2
(mouse)
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 OF EACH 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