Lab 8 quiz Flashcards
What are the six steps of western blotting? What is each one for
SETBPD
Sample preparation: Samples are boiled in the presence of mercaptoethanol, to completely denature the proteins in the sample
Electrophoresis: Proteins are separated by SDS-PAGE
Transfer: Proteins are transferred from polyacrylamide gel onto a nitrocellulose membrane
Blocking: Unoccupied binding sites are blocked by binding milk solution proteins.
Probing with Primary antibody: recognizes and bands specificallly to the protein of interest is incubated with the nitrocellulose membrane, unbound antibody is washed off
Detection: A tag is used, conjugated to a secondary that binds the primary
Describe sample preparation
SETBPD
Sample preparation: Samples are boiled in the presence of mercaptoethanol, to completely denature the proteins in the sample
Describe electrophoresis
Electrophoresis: Proteins are separated by SDS-PAGE
Describe transfer
Proteins are electrophoretically transferred from the polyacrylamide gel onto a nitrocellulose membrane.
Describe blocking
Blocking: Unoccupied binding sites on the membrane are blocked by enabling them to bind
proteins from a milk solution. This step prevents the nitrocellulose from non-specifically
binding additional proteins such as antibodies and, thus, decreases background signal
Describe probing with primary antibody
Probing with Primary antibody: An antibody that recognizes and binds specifically to the
protein of interest is incubated with the nitrocellulose membrane. After allowing the
antibody to bind to the protein on the membrane, unbound antibody is washed off the blot
describe detection
Detection: In order to detect the protein of interest, a tag such as a radioisotope or enzyme
is used. Sometimes the tag is conjugated to the primary antibody, but most often the tag is
conjugated to a secondary antibody that binds to the primary antibody as shown below.
Break down the detection step
- Probing with a secondary antibody: binds to the constant region of the primary, amplifies signal, can detect different primary antibodies
- Visualization: Enzyme or radioactive tag is used to bind the secondary antibody
Advantages and disadvantages of radioactive tag
Advantages:
blots can be “stripped” of one antibody and reprobed with another
Disadvantages:
Can be slow
Work with and dispose of radioactivity
Advantages and disadvantages of Alkaline phosphatase
advantages:
- obtain results relatively fast
- can watch bands appear and control exposure
- relatively inexpensive
Disadvantages:
- background is often higher than with other methods of detection
- cannot strip and reprobe blots
- bands tend to fade with time; so need to have results photographed
Advantages and disadvantages of horseradish peroxidase
Advantages:
- Very sensitive
- Obtain results very fast
- Low background
- Hard copy results on film
Disadvantages:
- Relatively expensive
What is an alkaline phosphatase
A chromogenic assays using CIP and NBT yielding insoluble precipitate on the membrane, Results are photographed
What is horseradish peroxidase
Conjugated secondary antibodies; a chemiluminescence reaction produces light which is detected by X-ray film
Why transfer proteins onto nitrocellulose rather than simply working directly with the original gel
A membrane is easier to handle and manipulate without it breaking
Low concentrations are more easily detected, because they are concentrate on the surface
staining and destaining is faster
The blot is convenient
What do methanol and SDS do in the transfer buffer
Methanol causes a reduction in pore size of the gel, increases binding capacity of nitrocellulose
SDS may improve elution of high molecular weight proteins
