What treatment did the CHO cells undergo in the 1st step of the experiment?
They experienced cold or heat shock stress.
What buffer was used to lyse the cells in the 2nd step?
RIPA buffer.
What was the purpose of the Bradford Assay performed in the 3rd step?
To determine the protein concentration of the samples.
How were the proteins separated in the 4th step?
By mixing lysates with Sample Buffer and electrophoresing them through a polyacrylamide gel.
Where were the proteins transferred to in the 5th step?
From the gel onto a piece of nitrocellulose membrane.
What is the primary goal of today’s Western blot procedure?
To identify a specific protein of interest and assess its expression levels.
What biological molecules does Western blotting utilize to detect proteins?
Antibodies.
What does “protein expression” assessment look for in this experiment?
Whether conditions caused up-regulation or down-regulation of a protein.
Which two experimental proteins might you be probing for today?
Hsp90 or Tubulin.
Why are you probing for the metabolic enzyme GAPDH?
It serves as a procedural check (loading control).
What is an antigen?
The specific target protein that an antibody binds to.
In an IgG antibody with a Y-shaped structure; what do the “branches” represent?
The variable regions which complement the shape of the antigen and allow binding.
Which part of the antibody is identical for all IgG-class antibodies from a single species?
The constant domain (the base of the Y).
What is the general method for creating Polyclonal antibodies?
Injecting a host animal with purified antigen and harvesting the antibodies directly from the animal’s blood.
What distinguishes Polyclonal antibodies regarding how they bind to the protein of interest?
They are a mixture of antibodies that bind to different epitopes on the same protein.
What specific cell type is created to produce Monoclonal antibodies?
A hybridoma (fused antibody-producing white blood cell and immortal cell).
How do Monoclonal antibodies differ from Polyclonal antibodies regarding specificity?
Monoclonal antibodies are specific for the exact same epitope on an antigen.
What is the primary advantage of using Monoclonal antibodies in research?
They are highly specific and less likely to randomly bind to other proteins (background noise).
What is the function of a “reporter molecule” conjugated to an antibody?
It allows for the visualization of the antibody binding to its antigen (via fluorescence
In an “Indirect Visualization” method; what does the Primary antibody bind to?
The specific protein of interest (antigen).
In an “Indirect Visualization” method; what does the Secondary antibody bind to?
The constant region of the Primary antibody.
Why must the Secondary antibody be raised in a different host species than the Primary?
An animal cannot produce antibodies against itself; a different host is needed to recognize the Primary host’s constant region as foreign.
If a Primary antibody is named “Rat anti-kinesin-2”; what does “Rat” refer to?
The host animal that produced the antibody.
If a Primary antibody is named “Rat anti-kinesin-2”; what does “kinesin-2” refer to?
The specific protein (antigen) the antibody recognizes.
