Western blotting utilizes _______
to identify ______
displayed by ____
bound to ____
- antibody “probes”
- specific antigenic epitopes
- the proteins
- a solid support
western blotting combines ______
achieved by _____
with _____
of _____
- the high resolution
- SDS-polyacrylamide gel electrophoresis
- the specificity
- immunodetection
Western blotting provides information about _______
As well as ______
- the presence and quantity of a protein in different cells or tissues
- the molecular weight of a polypeptide chain
There are no ______ and ____ problems for electrophoretic separation of proteins because the conditions are usually
- insolubility
- aggregation
- denaturing
What two elements denature the proteins
SDS and ß-mercaptoethanol
For electrophoresis the proteins in the sample are seperated by _______ on the basis of ______
- SDS-PAGE
- molecular weight
Proteins are electrophoretically transferred from the _______ onto a ________
Polyacrylamide gel
nitrocellulose membrane
Why transfer proteins onto nitrocellulose rather than simply working directly with the original gel
(reason 1)
(reason 2)
Why reason 2
- a membrane is much easier to handle and manipulate without it breaking
- low concentrations of samples are more easily detected
- they are not spread throughout the thickness of the gel, are concentrated at the surface on the membrane
to facilitate ___________ from the gel to the membrane and to promote their ___________
a buffer containing ______ and _____ is used
- the migration of proteins
- binding to the membrane
- methanol
- SDS
methanol causes a _______
can make it more difficult for ______
and increases the _________ of ______ for protein
general reduction in pore size
proteins to leave the gel
binding capacity
nitrocellulose
SDS may ____________ of high molecular weight proteins but may interfere with ___________
- improve the elution
- binding of the proteins to the membrane
Why are prestained molecular weight markers used when proteins are to be transferred to nitrocellulose in a western blot
The molecular weight markers are visible on the membrane
__________ on the membrane are blocked by enabling them to _________
- Unoccupied binding sites
- bind proteins from a milk solution
Blocking prevents __________; proteins such as ________, thus decreasing
- the nitrocellulose from non-specifically binding additional proteins
- antibodies
- background signal
Why is it important to wear gloves when handling nitrocellulose
Nitrocellulose binds proteins, when binding with milk solution, you know what the proteins are which you do not want to detect, if the membrane gets contaminated with proteins from your hand, then unknown proteins are bound to the membrane, which may create ambiguous results because you have no plans to not detect these proteins
Why is it important to wash unbound antibody off the blot
If the membrane is not washed there will be unbound primary antibody which the secondary antibody may bind to.
This binding however will be weak and nonspecific
In order to detect the protein of interest ____ such as a ______ or ______ is used
a tag
radioisotope
enzyme
protein binds to _______
primary antibody binds to ________
secondary antibody binds to _____________
nitrocellulose
protein
protein-primary antibody complex
The detection step
i)
ii)
- Probing with a secondary antibody
- Visualization
Probing with a secondary antibody
This antibody binds to the _____ of the primary antibody
It allows a ________ secondary antibody to be used to detect many __________ primary antibodies
It provide an ________________, since several secondary antibody molecules can bind to each primary antibody
- constant region
- common
- many different
- amplification of the signal
Visualization
radioactive tag - _______________
Advantage - ________________
Disadvantages - _________
- autoradiography can be used to detect the ring
- blots can be “stripped” of one antibody and reprobed with another
- can be slow to obtain results, have to work with and dispose of radioactivity
Visualization
Alkaline phosphatase - _____________
Advantages - _____________
Disadvantages - ______________
- a chromogeneic assay using BCIP and NBT yields an insoluble precipitate on a nitrocellulose membrane
- results are relatively fast, can watch bands appear and control exposure, relatively inexpensive
- background is often higher than with other methods of detection, cannot strip and reprobe blots, bands tend to fade with time, so must photograph
Visualization
Horseradish peroxidase - _________
Advantages - _________
disadvantages - __________
- conjugated secondary antibodies; a chemiluminescence reaction produces light, which is detected by X-ray film-exposure
- very sensitive, obtain results very fast, low background, hard copy results on film
- relatively expensive
incorrect assembly of the sandwhich could result in
failure of the proteins to transfer to the membrane
