More techniques—
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Nucleic acids, in general
are big moleculesseparate them in a
loose-pore matrix
(agarose)
Gels by themselves
tell you the size
and amounts of
nucleic acidscommonly used to
check PCR reactions,
RNA isolations,
etc.
Can also be used
analytically with
restriction digestsRFLPs
Ed Southern first transferred pattern of bands to a stabile
membrane (why would you do that?) –then incubated the
membrane with a radiolabeled specific probe-called a Southern
blot and it tells you:
•gene presence
•copy number
•differences among genomes
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If you separate
RNA instead of DNAthen transfer the
gel to a membrane and
incubate it with a radiolabeled probe-it’s called
a Northern blot
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Tells you
•size
•presence
•amount of mRNA
If you separate proteins, and then transfer them
to a blot-then incubate them with antibodiesthat’s a Western blot (immunoblot)
Tells you•size
•presence of protein
•amount of protein
Proteins almost always separated on acrylamide
gels
(resin based) instead of agarose gels (carbohydrate
based). Gives you much smaller pores and thus you
can resolve much smaller things (note: when
differences
in size of nucleic acid strands is small-researchers
also
use acrylamide instead of agarose—e.g. sequencing)
WATCH WESTERN BLOT ANIMATION
ProteomicsStudy of a organism’s proteome- 2-d electrophoresis common
RNAse Protection AssayTells you
•presence
•can be more sensitive than Northern
•can detect very small differences in mRNA
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And a BAZILLION more techniques-but this should at least
give you a vocabulary. Let’s look at some real-life examples
of how these are applied…
Do Antarctic fish have
myoglobin in their hearts?
If not, have they lost the
ability to make it?
How would you approach this
problem?
Thousands of individual
proteins in the cell. We
know that myoglobin is
not associated with
organelles, cytoskeleton,
or plasma membrane-so
get rid of those with
differential
centrifugation
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Separate proteins in
cytosol by sizeblot to membraneincubate blot with
antibodies against
myoglobin.
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If you don’t see anythingcan you say for sure they
don’t have it?
Why not?
Family Channichthyidae
Chaenocephalus aceratus
Yes gene for Mb
No mRNA
No protein
Champsocephalus gunnari
Yes gene
Yes mRNA
No protein
Chionodraco rastrospinosus
Yes gene
Yes mRNA
Yes protein
Group Exercise 3
In groups of 4-answer these questions-turn in one group paper.
A) There is such a thing as a SouthWestern blot. What do you
think it is, how would you do it, and what would you be looking
for?
B) You are looking for evidence of a protein called ubiquitin
in grasshoppers. Devise a strategy to prove that it exists. You
want more than one line of evidence.