LEC 3 Flashcards
Agarose gel structure?
Tiny pores= mesh. When a potential difference is applied across the gel, the mesh impedes the progression of molecules (smaller molecules impede less)
Loading dye purpose in electrophoresis
Physically track the molecules
How to produce agarose gel?
Heat up agarose to at least 60 degrees (liquid form) pour into mould (with comb in place) to form the wells
Comb used to form?
wells
DNA is charged?
NEGATIVELY therefore moves towards the positive anode
Agarose gel is used to analyse what types of genomes?
medium sized gels
Polyacrylamide
gel used on smaller genomes (as can detect small differences in fragment size)
Production of polyacrylamide? (chemically)
Acrylamide+ methylene biacrylamide= polyacrylamide (cross linked).
In forming the gel on a plate (polyacrylamide)
Pour onto a plate (with a spacer between to create a gap) overlay with water to stop oxidation of polyacrylamide.
Two types of gel stain’s (DNA)
ETHIDIUM bromide (not as safe, cheaper) and SYBR GOLD (safer, more expensive, and detects small volumes of DNA)
Molecular marker use?
as a comparison: what fragments are present
gel stains (proteins)
coomassie blue (safer) and silver (not as safe but more sensitive to smaller volumes of protein)
Denaturing form?
chemical agents to break interactions within the conformation
Native form?
natural form
Foramide/ SDS
a chemical agent which breaks hydrogen bonds to linearise the protein (denatured)
when is the denaturing form relevant to use
when we are only interested in its size rather than conformation.
southern blotting? (not method, background)
ED southern 1975.
previous use: DNA
now: RNA
Method: southern blotting?
- Agarose gel
- Load DNA samples
- Lane for molecular marker
- Run gel
- Blotting: nitrocellulose membrane, paper towel and a weight.
- nitrocellulose has a high affinity for proteins
- Gel> membrane
- Remove nitrocellulose membrane, mix with a price which is complementary to the fragment of interest
- wash to remove the unbound probe
- Expose to xray film and if the fragment of interest is present then it will show up on the audio radiogram
Northern blotting used with?
RNA
characteristics of Northern blotting ?
quick and easy
To find the vol of protein expressed in particular places
method of Northern blotting
- Extract RNA, isolate, purify.
- isolation: magnetic bead s in which have a series of Thymine nucleotide bases attached- oligodT dynabead
- these attach themselves to poly a tails of mRNA = dimer beads
- run mRNA like blotting
- Apply complementary RNA probe again to isolate
- Purified mRNA is formamide denatured (linearised)
- run on gel
- Radioactively labelled with a probe
- We can now see by gel electrophoresis by the sizes of the bands how well a particular gene is expressed in tissues
Western blotting- antibodies method?
-Antibodies are raised against a protein of interest.
- Proteins are electrophoretically transferred to the nitrocellulose layer from the gel
- SDS mutant used to denature protein
- place in a tank with a buffer and apply a potential difference
- Antibody has a great affinity for the nitrocellulose membrane.
- Chemlucinet enzyme flashes light when reaction takes place (s>p)
- bands show up if light emitted
- Expression of genes detected
fc 8 to continue
difference between western blotting antibodies and eastern blotting method?
In eastern blotting- proteins migrating to nitrocelluose by capillary action
in western blotting- proteins are electrophoretically transferred to the nitrocellulose layer
what type of enzyme is used in western blotting?
chemilucent
