Lab 5 Flashcards
electrophoresis
is used to visualize the movement of charged molecules in a electric field.
molecules are separated based on mobility through the gel
larger molecules tend to move more slowly
gels are made using either agarose or polyacrylamide
Agarose VS PA
AGAROSE
Easier to prepare
Can be used on large proteins and DNA
Low resolution/greater range
POLYACRYLAMIDE Difficult to prepare Neurotoxin Fine resolution/lower range Uniform Pore size
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis,
used widely in biochemistry
separate proteins according to their size
smaller proteins move faster
larger proteins move slower
Sodium-Dodecyl-Sulfate
SDS
anionic detergent, linerizes proteins, puts a negative charge on proteins
what does adding SDS do?
the protein becomes denatured by SDS (reduced to its primary structure amino acid sequence) and as a result has been linearized
SDS binds to proteins in a constant mass ratio of 1.4:1, so that the total amount of detergent bound is directly proportional to the molecular weight of the protein.
PAGE
If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size.
However, if the proteins are put into an environment that will allow different sized proteins to move at different rates they will separate by size.
polyacrylamide
Small molecules move through the polyacrylamide faster than big molecules.
Big molecules stays near the well.
Two different gels stacked
- Stacking gel
- Running gel
stacking gel
To obtain optimal resolution of proteins.
Stacking gel is on top of running gel.
Lower concentration of acrylamide (large pore size) (4%). Lower pH (~6.8).
Different ionic content.(chlorine ions)
Concentrates the proteins in a lane into a tight band before they enter the running gel.
Results in better separated protein bands.
Glycine slower than proteins, helps concentrating.
running gel
To separate proteins according to their sizes
Higher pH (~8.8).
Acrylamide concentration between 12-18%.
Different concentration,different resolution.
Creates smaller pores and a sieving effect.
Glycine faster than proteins.
protein migration graph:
logMr on y
relative migration on X
protein size
Protein size measured in kDa
Dalton = standard unit used to indicate mass on an atomic or molecular scale.
Gel staining:
After the proteins have been separated by electrophoresis gel needs to be stained.
Goal: stain proteins but not the polyacrylamide.
Most widely used: Coomassie Blue R250
In addition to binding hydrophobic regions, R-250 Interacts with carbon-nitrogen backbone and binds to most proteins uniformly.
destaining
Staining will leave some residue on gel, therefore it needs to be destained to show only proteins.
A solution of methanol and acetic acid is used to dissolve the stain on the gel.
Takes about 1-2 days.
Solution needs to be changed frequently.
What did we do in lab?
You will be mixing 2.5ul of Loading buffer with 7.5ul of Protein sample(BSA)
We incubate for 5-10 mins at 70-95C respectively
Load sample into gel and apply 150V for 40 minutes
Stain & destain
loading buffers
SDS
Denatures proteins and provides negative charge
β‑mercaptoethanol
Denatures proteins
Reducing agent
Removes disulfide bonds
Glycerol
Weighs down sample
Bromophenol Blue
Tracking Dye
