MCB Lab Practical - Separation of Proteins Part II & III

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?

(-) top
(+) bottom

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

western blotting

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 ?

indirect immunoblot

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.
Chromogenic substrat

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