lab 8

Question 1

Western blotting utilizes _______
to identify ______
displayed by ____
bound to ____

Answer 1

• antibody “probes”
• specific antigenic epitopes
• the proteins
• a solid support

Question 2

western blotting combines ______
achieved by _____
with _____
of _____

Answer 2

• the high resolution
• SDS-polyacrylamide gel electrophoresis
• the specificity
• immunodetection

Question 3

Western blotting provides information about _______

As well as ______

Answer 3

• the presence and quantity of a protein in different cells or tissues
• the molecular weight of a polypeptide chain

Question 4

There are no ______ and ____ problems for electrophoretic separation of proteins because the conditions are usually

Answer 4

• insolubility
• aggregation
• denaturing

Question 5

What two elements denature the proteins

Answer 5

SDS and ß-mercaptoethanol

Question 6

For electrophoresis the proteins in the sample are seperated by _______ on the basis of ______

Answer 6

• SDS-PAGE

- molecular weight

Question 7

Proteins are electrophoretically transferred from the _______ onto a ________

Answer 7

Polyacrylamide gel

nitrocellulose membrane

Question 8

Why transfer proteins onto nitrocellulose rather than simply working directly with the original gel
(reason 1)
(reason 2)
Why reason 2

Answer 8

• 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

Question 9

to facilitate ___________ from the gel to the membrane and to promote their ___________
a buffer containing ______ and _____ is used

Answer 9

• the migration of proteins
• binding to the membrane
• methanol
• SDS

Question 10

methanol causes a _______
can make it more difficult for ______
and increases the _________ of ______ for protein

Answer 10

general reduction in pore size
proteins to leave the gel
binding capacity
nitrocellulose

Question 11

SDS may ____________ of high molecular weight proteins but may interfere with ___________

Answer 11

• improve the elution

- binding of the proteins to the membrane

Question 12

Why are prestained molecular weight markers used when proteins are to be transferred to nitrocellulose in a western blot

Answer 12

The molecular weight markers are visible on the membrane

Question 13

__________ on the membrane are blocked by enabling them to _________

Answer 13

• Unoccupied binding sites

- bind proteins from a milk solution

Question 14

Blocking prevents __________; proteins such as ________, thus decreasing

Answer 14

• the nitrocellulose from non-specifically binding additional proteins
• antibodies
• background signal

Question 15

Why is it important to wear gloves when handling nitrocellulose

Answer 15

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

Question 16

Why is it important to wash unbound antibody off the blot

Answer 16

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

Question 17

In order to detect the protein of interest ____ such as a ______ or ______ is used

Answer 17

a tag
radioisotope
enzyme

Question 18

protein binds to _______
primary antibody binds to ________
secondary antibody binds to _____________

Answer 18

nitrocellulose
protein
protein-primary antibody complex

Question 19

The detection step
i)
ii)

Answer 19

• Probing with a secondary antibody

- Visualization

Question 20

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

Answer 20

• constant region
• common
• many different
• amplification of the signal

Question 21

Visualization
radioactive tag - _______________

Advantage - ________________
Disadvantages - _________

Answer 21

• 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

Question 22

Visualization
Alkaline phosphatase - _____________
Advantages - _____________
Disadvantages - ______________

Answer 22

• 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

Question 23

Visualization
Horseradish peroxidase - _________
Advantages - _________
disadvantages - __________

Answer 23

• 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

Question 24

incorrect assembly of the sandwhich could result in

Answer 24

failure of the proteins to transfer to the membrane