Antibodies and Immunodetection

Question 1

What methods in imumunodetection can be used to detect protein?

Answer 1

Immuno dot blots, western analysis, immunocytochemistry and immunoprecipitation.

Question 2

What method can be used in vivo to detect protein/DNA interactions in vivo?

Answer 2

Chromatin precipitation

Question 3

What method can be used to detect the rate of synthesis?

Answer 3

Pulse chase

Question 4

What method can be used to measure synthesis in vitro?

Answer 4

Wheat germ extract and GST pull down experiments

Question 5

What is the difference between polyclonal and monoclonal antibodies?

Answer 5

Polyclonal are a population of antibodies that recognise different regions and have different affinities for your protein whereas monoclonal is a single antibody species produced using a clonal cell line called a hybridoma.

Question 6

How are monoclonal antibodies produced?

Answer 6

Immunisation of a mouse using an antigen, the immune cells are then isolated as antibody-forming cells and fused with tumour cells to form a hybridoma. The hybridoma is screened for production of desired antibody and cloned. Monoclonal antibodies are produced through clonal expansion.

Question 7

What methods are their to directly label antibodies?

Answer 7

Radioactively or fluorescently

Question 8

What are the advantages of directly labelling antibodies?

Answer 8

Convenient and simple

Question 9

What are the disadvantages of directly labelling antibodies?

Answer 9

Health risk, potentially poor signal and can be costly.

Question 10

What methods are their to indirectly label antibodies?

Answer 10

Use a secondary antibody that recognises the constant region of a primary antibody, the secondary anybody is usually linked to a fluorophor such as FITC (green), rhodamine (red) or enzyme.

Question 11

What enzymes can be linked to the secondary antibody in indirect labelling?

Answer 11

Horseradish peroxidase and alkaline phosphatase.

Question 12

What are the advantages of indirectly labelling antibodies?

Answer 12

Can amplify a signal, therefore very sensitive, can use the same secondary antibody with multiple primary antibodies, therefore cost effective.

Question 13

What are the disadvantages of indirectly labelling antibodies?

Answer 13

Have to perform two rounds of detection in protocol.

Question 14

How are immuno dot blots carried out?

Answer 14

Protein extract is spotted on a nitrocellulose membrane and it is analysed using an antibody.

Question 15

Describe the steps in performing a western blot?

Answer 15

1) Extract protein form cells/tissues and carry out electrophoresis- separation based on size.
2) Transfer to nitrocellulose membrane
3) Calculate Mr from distance moved
4) Incubate with specific antibody, wash off
5) Visualise where the antibody was using, most commonly, secondary antibody.

Question 16

What is used in SDS PAGE and why?

Answer 16

Negatively charged SDS to keep the protein denatured and eliminate the intrinsic charge to allow the protein to migrate through the gel solely by charge. Add beta-mercaptoethanol or dithiothreitol to keep the protein denatured and remove disulphides bridges between cysteine residues.

Question 17

How is the nitrocellulose membrane first prepared?

Answer 17

It is blocked, to prevent non-specific binding to the membrane, by any cheap protein eg. Milk marvel powder

Question 18

How accurate is it calculating Mr from migration through and SDS PAGE?

Answer 18

Glycosylated proteins show larger Mr due to bulkiness but otherwise accurate.

Question 19

What is the most popular method of detection using secondary antibody?

Answer 19

Horse radish peroxidase.

Question 20

How does horse radish peroxidase work?

Answer 20

Catalyses the release of light upon the reaction with luminol/H2O2 and can be used to determine where the antibody is bound.

Question 21

What are protein A and G?

Answer 21

Protein G is a cell wall protein isolated from Type G Streptococci and protein A is derived from Staph A. Both bind in the constant region of IgG with high affinity and can be used to facilitate the purification and recover of either poly or monoclonal antibodies.

Question 22

What can protein A and G be linked to?

Answer 22

Agarose, magnetic beads and solid supports.

Question 23

Describe the steps of immunoprecipitation?

Answer 23

1) Add antibody to cell lysate
2) Add protein G agarose
3) Spin to precipitate
4) Remove supernatant
5) Elute with SDS

Question 24

What are the uses of immunoprecipitation?

Answer 24

To look at proteins expressed at low levels, post translational modifications, to identify components of protein complexes and to identify whether protein interact.

Question 25

What can co-immunoprecipitation be used for?

Answer 25

Determining protein interactions and identifying proteins in complexes

Question 26

Describe the steps of co-immunoprecipitation?

Answer 26

1) Identify and extract the cell nucleus donating the two interacting proteins of interest.
2) Add antibody directed against one of the proteins of interest.
3) Add antibody bidding beads.
4) Immunoprecipitate the protein of interest.
5) Wash and collect the immunoprecipitated proteins
6) Western blot analysis of the immunoprecipitated proteins using an antibody directed against the second protein of interest.
If they interact they will be identified.

Question 27

What is the shorthand for chromatin immunoprecipitation?

Answer 27

ChIP

Question 28

What does chromatin immunoprecipitation determine?

Answer 28

The DNA sequences that are bound by specific proteins or modified Histones.

Question 29

In ChIP what do you raise antibodies against and use primers against?

Answer 29

Antibodies: Transcription factors, chromatin associated proteins or modified histones
Primes: use them against a specific gene in the PCR reaction to determine if that region is bound by the protein of interest

Question 30

How can ChIP products be analysed using:

a) PCR?
b) qPCR?
c) microarray?
d) ChIPSeq?

Answer 30

a) whether the protein binds
b) quantify the level bound
c) if the protein binds to a large number of different sequences and it’s affinity
d) determine all of the sequences the protein binds

Question 31

How does pulse chase measure ether rate of synthesis/ degradation of protein?

Answer 31

Incubate cells for a set time with 35S Met for set time- pulse
Incubate cells with excess unlabelled Met- chase
And take protein extracts at several time points, immunoprecipitate with antibody, run on a SDS-PAGE and quantify the 35S labeled protein.

Question 32

How can you measure protein synthesis?

Answer 32

Use wheat germ or reticulocytes, lyse them and spin to remove nuclei, mitochondria and plastids, use of purify further.

Eg. As prey in GST pull down experiments.

Question 33

What tools are required for immunodetection?

Answer 33

Antibodies and labels to visualise proteins.