Week 6 - Electrophoresis Flashcards
How to identify heavy molecules?
moves slower
How to identify small molecules?
moves more fast and are more charged
Ohm’s law
V/I = R
V=voltage I=current R=resistance
V/I = R
V=voltage I=current R=resistance
What is the medium used in electrophoresis?
agarose, polyacrylamide
What is the capillary used in electrophoresis?
pore size, ionic strength
What determines mobility in electrophorsis?
speed of migration
What happens if we apply constant voltage?
current will eventually increase
because resistance of medium is decreasing
What is agarose gel used for?
DNA
What is polyacrylamide gel used for?
high resolution, proteins and smaller
nucleic acid.
Whats the difference between polyacrylamide and agarose?
smaller pore size of polyacrylamide compared to agarose
- better separation of small molecules
How can capillary achieve high resolution separation?
narrow capillary with buffer
APPLICATIONS OF ELECTROPHORESIS
DNA fingerprinting
DNA sequencing
Protein analysis
Clinical diagnosis – serum proteins, biomarkers
Pharmaceutical development – characterisation of biologics e.g.
antibodies, vaccines
What does higher concentration of agarose mean?
smaller mesh
- more agarose = smaller the sieve
agarose structure
Horizontal
Native structure
What is commonly used at a loading dye in agarose gel?
bromophenol blue, xylene cyano
made also with glycerol
What type of staining is used in agarose gel?
Ethidium bromide, non-toxic fluorescent stains
Ethidium bromide
carcinogenic
What can be determined from agarose gl electrophoresis?
- Size determination
- DNA or RNA extraction
- DNA and RNA
Feature of agarose gel
we can physically cut out band and re-examine
What is a southern blot?
a laboratory technique used to detect specific DNA sequences in a DNA sample
- the samples are chewed up using DNA restriction enzymes normally in intracellular membrane
How to set up a southern blot?
use paper to blot and wick up buffer
start to more liquid/buffer up towards the well so we have gel on top
transfer the membrane on top
put something heavy on it
= we have cellulose membrane with DNA transferred
How do you use a radio cable P32 probe?
incubate membrane with probe
wash off excess
all of prob hydrolyse to particular fragment of DNA that we transferred to membrane
expose to x-ray film
shows restriction image
What does SDS-PAGE involve?
removing tertiary structure
denatured protein (SDS, β mercaptoethanol, boiling)
discontinuous buffer system
SDS
sodium dodecyl sulphate
PAGE
polyacrylamide gel electrophoresis
What gel does SDS-PAGE use?
5%-20% polyacrylamide
What way do samples run on SDS-PAGE?
Vertical
Where does SDS-PAGE migrate to?
towards positive electrode
What is the mythology of SDS?
disrupted bonds
What type of reaction is making polyarcylamines?
polymerisation reaction
What is the loading dye for SDS-PAGE?
Bromophenol blue
What is the staining for SDS-PAGE?
Coomassie blue, non-
toxic fluorescent stains
What does SDS-PAGE determine?
size and protein extraction
Why does DBS have different pH between stacking gel and resolving gel?
allows to load off samples and all out samples are ready and lined up so they can migrate
What is the buffer placement like in DBS?
buffer at top and bottom
What are the other types of gels for SDS?
Non-denaturing gels/native gels – separation, identification of protein of interest by substrate
Gradient gels – for more complex mixtures, separation of similar Mr values
Isoelectric focussing gels – based on pI (iso-electric points)
2D PAGE gels
What does non-denaturing gels/native gels allow?
separation, identification of protein of interest by substrate
What do gradient gels allow?
for more complex mixtures, separation of similar Mr values
What are Isoelectric focussing gels based on?
based on pI (iso-electric points)
What are the advantages of gradient gel?
more specific gel creation with different pore sizes and concentration
and smaller molecules are going to percolate further
Describe the molecule distribution in gradient gels.
smaller molecules are going to percolate further
larger molecules are going to be stuck at the top because they can’t pass through the sieve
different sizes of molecules are going down
What is immunoblotting/western blotting?
Transfer of products to a solid stable membrane – nitrocellulose
Further investigate products – antibodies
Process of protein: antibody detection
- coat surface with sample (antigen)
- block unoccupied sites with non-specific proteins
- incubate with primary antibodies against specific antigens
- incubate with primary antibody against specific antigen
- add substrate
- formation of coloured products indicates presence of specific antigen
What does isoelectric focussing tell us?
isoelectric points of protein
Process of isoelectric focussing
a protein sample may be applied to one end of a gel strip with an immobilised pH gradient.
or a protein sample in a solution of ampholtes maybe used to rehydrate a dehydrated gel strip
an electric field is applied
after staining, proteins are shown to be distributed along pH gradient according to their pI values
What happened if the spot is at the positive side in the isoelectric focussing?
lower pI - lower pH
What happened if the spot is at the negative side in the isoelectric focussing?
higher pI and higher pH
What happens if the pH is the same as pI?
netcharge = 0
What does 2D electrophoresis allow?
allows a 2 way separation
What are the two separation proteins in the two dimensions?
1D
= using isoelectric focusing
2D
= SDS-PAGE gel
What does capillary electrophoresis allow?
checking purity
