What type of gel was used in this experiment?
What is the distribution of polyacrylamide down the gel?
low % (top) -> high % (bottom)
small proteins travel shorter/further distances than large proteins
SDS-PAGE separates proteins based on ?
differences of charge and size/shape
What does SDS do?
coats protein backbone with a (-) charge & linearizes the protein, removing 2? structure
What charged electrodes are on each end of the SDS-PAGE gel?
What type of stain is used after SDS-PAGE is run on the gel?
Coomassie Blue stain
Allows you to identify proteins of interest with specific antibodies
Which is Y-shaped: antibody or antigen?
How do you balance a centrifuge?
-Make sure the tubes are logistically balanced (ie opposite of each other)
-Make sure the balancing tubes have the same VOLUME of liquid as the sample tube
What type of graph paper did we use to plot MW (y) vs Rf (x)?
Western blot is also called ?
How does a western blot work?
Membrane contains proteins that act as antigens
1? antibody is run across the membrane and attaches to proteins (antigens) of interest
2? antibody against the first antibody is run across the gel. 2? antibody is attached to an enzyme.
In what region of the 1? antibody does an antigen binds?
In what region of the antibody does the 2? antibody bind?
What does a Pierce 660nm assay do?
AKA "Protein Quantification Assay"
Relates the concentration of protein to color change that is monitored by the spectrophotometer
T/F: Presence of certain detergents/reducing agents can interfere with Pierce 660nm assay.
How does color change occur in Pierce 660nm Assay?
The assay uses a dye + a transition metal that forms a complex.
Dye-metal complex has a reddish-brown color that changes to green upon binding to a protein
Color change shifts the absorption of the sample, which can be analyzed to determine concentration
what was the blocking solution and why was it used?
Blocking Solution = Milk
Milk contains casein protein that will take up spots on the membrane and stop the reaction
In abs vs. concentration standard curve, what type of paper do you use and what is each axis?
Non-log graphing paper
Y-axis = absorbance at __nm
X-axis = concentration
When plotting MW vs Rf what are the axes and what paper is used?
Semi-Log Graph paper
Y-axis = MW (g/mole)
X-axis = Rf Value