Cell Biology Lab 9: Western Blotting

Western blotting

procedure that uses labeled antibodies to detect specific antigens in a mixture of proteins separated according to their molecular weight

How do the primary antibody in a western blot technique interact with the secondary antibody directed against it

The secondary antibody is directed against the constant region of the primary antibody

The purpose the secondary antibody serves in Western Blotting

It serves not only as a carrier of the label but it is also a mechanism to amplify the emitted signals because as many of these kinds of antibodies can theoretically bid simultaneously to the primary antibody

What is measured from the secondary antibody after binding to the primary antibody

The signal is measured

What is the signal emitted by the secondary antibody proportional to

The quantity of protein of interest present on the membrane

What is the purpose of the blocking step in Western Blotting

It reduces the number of nonspecific protein interactions between the membrane and the antibody. Once all the proteins of the dilute solution has attached to the membrane, there is no room on the membrane for the antibody to attach other than the binding sites of the specific target protein

What can be used for blocking in Western Blotting

Dilute solutions like 3-5% Bovine Serum Albumin (BSA) or non-fat dry milk in TBST (Wash buffer)

How does dilute solutions attach to the membrane in blocking during Western Blotting

The protein in the solution attaches to the membrane in all places where the target proteins have not attached.

Polyclonal antibodies

Tend to be more sensitive. They are less specific than monoclonal antibodies. They are usually chosen for their relatively lower price as they are less time-consuming to produce

Monoclonal antibodies

Tend to be more specific but less sensitive. Bind to only one epitope and ae typically highly specific, pure and consistent in performance, and generally give rise to low backgroudnd

What can lead to poor results based on the amount of primary antibody present?

High primary antibody concentration is a common reason for poor results

How does a high primary antibody concentration affect the results of Western Blotting??

High primary antibody concentration can result in there being a high background or non-specific bands

What are the most common classes of secondary antibodies and why?

anti-mouse IgG and anti-rabbit IgG are the most popular due to many primary antibodies being produce in mouse or rabbit host species

What does the enzyme horseradish peroxidase (HRP) do

It cleaves a chemiluminescent agent, and the reaction product produces luminescence in proportion to the amount of protein

What kind of form can secondary antibodies be expressed in

They can be expressed in conjugated forms

What is TBS-T (wash buffer) a mixture of

It is a mixture of Tris-Buffered Saline and Tween 20

Why is TBS-T used in Western Blotting

It is a buffer that is used to wash the PVDF for nitrocellulose membrane in Western Blotting

Tween 20

A relatively non-toxic detergent that helps wash the blot and remove any proteins that are non-specifically bound, therefore decreasing the background signal

How are the results of Western Blotting detected?

Using chemiluminescence (ECL)

How is chemiluminescence used in Western Blotting

The light emitted in most luminescent assays is of low intensity and of short duration. With this, we use an enhanced version of the chemiluminescence reaction. This involves performing the reaction with luminol in the presence of modified phenols, which greatly enhances light emission

What is used during Western Blotting to create an image of the antibodies bound to the blot

A sensitive sheet of photographic film is placed against the membrane, and exposure to the light from the reaction creates an image of the antibodies

What biological activity does Staurosporine have

It has the biological activity of inhibiting protein kinases through the prevention of ATP binding to the kinase.

How does Staurosporine induce apoptosis

It is able to activate caspase-3

What does Caspase-3 do

It is responsible for chromatin condensation and DNA fragmentation in apoptosis

Loading Control

Used in western blot to make sure same amount of protein is loaded for each run. It is also used for comparing the amount of protein loaded per lane, to ensure the total protein was transferred correctly, and that the reagents in Western Blotting are functioning normally

What were the primary and secondary antibodies prepared with in our lab

TBS0T with 5% milk

What happens after the membrane was left to sit in the blocking reagent (the milk in our lab)

It was rinsed with TBS-T 4 times for 2 minutes each