L5 and L6 Detection of biological molecules, specific proteins, and intermolecular interactions

Question 1

Describe how spectroscopy works

Answer 1

optical absorbance is measured as light passes through a sample containing the dye of interest and can then be measured using beers law

Question 2

What is the maximal absorbance of proteins

Answer 2

maxima at 280nm

Question 3

What is the maximal absorbance of nucleic acids (DNA and RNA)

Answer 3

maxima at 260nm

Question 4

Explain why proteins have very different extinction coefficients where as nucleic acids have similar extinction coefficients

Answer 4

The absorbance of proteins is due to the amount of tryptophan in a peptide chain whereas in DNA and RNA the absorbance is due to the aromatic bases which have similar absorances

Question 5

How can the sensitivity in the detection of proteins/nucleic acids be increased?

Answer 5

by binding them to molecules that fluoresce or absorb specific wavelengths of light (coomassie or fluorescent dyes)

Question 6

How specific is the binding of proteins to Coomassie dyes

Answer 6

nonspecific bind (used to quantify protein concentrations)

Question 7

Ethidium bromide binds to which bases pairs

Answer 7

intercalates between hydrophobic base pairs

Question 8

Benefit of using labeled tracers

Answer 8

they allow very small amounts of substances to be detected

Question 9

What are examples of some of the applications of labeled tracers

Answer 9

autoradiography, phosphorimaging, liquid scintillation counting

Question 10

What are examples of some of the applications of non-radioactive tracers

Answer 10

fluorescent microscopy, enzyme coupled chemiluminescence

Question 11

What is standard PCR used for and what is a disadvantage of the method

Answer 11

it is used as an endpoint analysis, however it does not allow you to be able to quantify the amount of copies before and after the reactions

Question 12

Describe how SYBR green works in PCR

Answer 12

by binding to double stranded DNA; During denaturation sybr green is released from a template dsDNA, following polymerization the dye binds to newly synthesized dsDNA

Question 13

What is real time PCR good for

Answer 13

quantification of the starting template

Question 14

What is the relationship between threshold and template amount in rtPRC

Answer 14

A threshold is set in the exponential phase; number of cycles it takes to reach Ct is inversely correlated to the starting template amount

Question 15

what are activity assays good for?

Answer 15

they provide a function readout of the activity of an enzyme or product even if a sample is impure; the product of a reaction may be easier to detect than the enzyme (product accumulate after round of catalysis)

Question 16

What components are needed to look for the activity of DNA pol

Answer 16

dNTPs (radiolabeled), Mg2+ buffer, primers, template DNA

Question 17

What is an epitope

Answer 17

the region of a protein that is recognized by the antibodies

Question 18

how do western blots work

Answer 18

the use antibodies to detect specific proteins in a mixture

Question 19

primary antibodies in WB

Answer 19

recognize a target protein (must be diverse set of proteins); examples; mouse anti-GFP antibody, mouse anti-H2a antibody

Question 20

secondary antibodies in WB

Answer 20

recognize any antibody that is produced by a particular species (limited)

Question 21

What steps are involved in WB

Answer 21

SDS-page, them a transfer of proteins from the gel to a membrane, followed by reactions with the primary and secondary antibodies, and then detection of the secondary antibody by radiography, fluorescence, chemiluminescence,etc.

Question 22

Why do we use protein tags

Answer 22

its not always possible to make Ab for proteins, so tags are added to the N or C terminus of proteins and Ab can be generated for the tags

Question 23

Define co-purification

Answer 23

purification of a molecule that interacts and is bonded with another molecule

Question 23

which tags facilitate purification

Answer 23

6xhis, GST, MBP

Question 24

Define Co-immunopurification

Answer 24

the use of antibodies to precipitate a protein and it’s interacting molecules

Question 24

What steps are involved in Co-IP

Answer 24

a matrix is treated with a primary antibody, a secondary antibody bound to agarose or magnetic beads are added, the samples are then washed, followed by elution to isolate the components

Question 25

Applications of Co-IP

Answer 25

detect interaction between molecules (after crosslinking), identify which regions a protein will bind to chromatin (ChIP), or identify which RNA is bound to a protein (CLIP)

Question 26

After ChIP

Answer 26

DNA/RNA will need to be amplified (determined by some types of sequencing methods)

Question 27

Describe the step involved in ChIP

Answer 27

cells are treated with formaldehyde (crosslink), followed by a digestion or sonication, then antibodies specific to the protein are added to precipitate, then crosslinking is reversed and components are isolated

Question 28

uses of EMSA

Answer 28

use to measure binding affinity, and requires non-denature conditions; mobility depends on the charge, shape, and size

Question 29

what is DNAase foot printing used for

Answer 29

to ID the region of where a protein bind to DNA

Question 30

How does DNA footprinting work

Answer 30

By labeling one end of a DNA molecule, a nuclease cuts non-specifically but does not cut where a protein is bound; it can distinguish where a protein is by cutting any radiolabeled end of a DNA molecule; results are analyzed on a denaturing page