Supplementary practical - SDS-PAGE Flashcards
on the polyacrylamide gel, mobility of molecules is directly proportional to what?
log10 of the mass of the protein
heavier proteins will be closer to the loading well - lighter ones move further down the gel
which specific cellular compartment was targeted by the increased expression of protein via the insertion of a novel gene?
periplasm
what does the beer-lambert law describe with regards to path length, conc and absorbance
what shape will this law take on a graph? which coefficient is the gradient
as the path length or conc increases, the absorption increases
straight line, extincion coefficient is gradient
the unknown goes on the x axis. what is the unknown for this practical?
protein concentration
what does a bradford assay measure? is it colorimetric?
the presence of basic amino acids (his, lys, arg), which form a protein-dye complex
yes it is colorimetric
which compound binds to the lac repressor, releasing it, switching on T7, causing transcription of proteins?
IPTG
what is the promoter of PET21a called? is PET21a a lac repressor or promoter?
T7
repressor
what is the pH of the upper stacking gel in SDS PAGE?
does it have small or large pores?
does it have a high or low percentage of acrylamide?
6.8
large pores due to low % acrylamide
what is the pH of the lower resolving/main gel in SDS PAGE?
does it have small or large pores?
does it have a high or low percentage of acrylamide?
8.8
small pores due to high % acrylamide
what are the main carriers of the current in the gel in SDS PAGE?
proteins with SDS
what is the mobile anion in both gels of SDS PAGE? which molecule has no charge in the upper stacking gel?
CL-
glycine
what is the function of Tris buffer?
keep pH constant
what is the function of glycerol in this practical?
provides weight to solution so material sits at bottom of wells
what is SDS? what does it do to cells and proteins? how?
what does it add specifically to proteins?
a detergent which lyses cells and solubilises/denatures the proteins by breaking S=S bonds
also adds a negative charge to proteins so it runs down the gel to anode
which reagent gives colour to the reaction so you can see how the gel is running?
bromophenol blue
how do you calculate retardation factor? which axis would it be plotted on against log10 MW?
distance moved by protein divided by distance moved by dye front
plotted on x axis
what can IPTG, mimicing allolactose, induce, when cells are provided with lactose?
the lac operon
transcription of the lac structural genes only occurs when ……. is present and ……. is absent
lactose present
glucose absent
what does the lac repressor bind to?
the lac operator
what does SDS coat denatured proteins evenly in?
negative charge
after proteins have been denatured and coated evenly in negative charge by SDS, what does this prevent them from doing?
folding
on an acrylamide gel, how would you measure the distance migrated by an unknown protein?
measure from the TOP of the gel down to the middle of the unknown protein band. Then you would measure the unknown band against the nearest MW standard.
on an acrylamide gel, how would you measure the distance migrated by the dye front?
using a ruler you would measure from the dye front’s starting line at the bottom of the gel, up to the top of the gel where it has stopped moving. measure in mm
how would you calculate the Rf value of an unknown protein you have on your gel?
distance moved by protein (mm) divided by the distance moved by the dye front (mm)
how would you estimate the MW of an unknown protein from a drawn standard curve (log MW against Rf value)?
it has an Rf value of 0.55
on the x axis (Rf value), find 0.55.
plot a line up to the standard curve, then plot another line across to the y axis to read off the log MW value
then to de log the value, you would do 10^ of whatever the log MW value is
a standard curve of logMW (y axis) against Rf value (x axis) should have which shape? which direction should it be going?
it is a straight line coming from top left to bottom right of graph
what is the ideal voltage to run your SDS-PAGE electrophoresis at?
40-180 V
how should the gel be trimmed for analysis after the dyes have run? what shape should it have?
should be rectangular in shape
should be trimmed so that all bands are contained, but none of the wells (cut off the wells)
when staining the gel, how many rounds of staining are done, followed by how many rounds of de-staining?
one round of staining followed by 2 rounds of destaining
when plotting. standard curve for Rf values and logMW values, what would you put on the x and then the y axis?
x axis = Rf values (calculated from gel)
y axis = logMW values
