Research

Question 1

In vitro assays

Answer 1

used to measure the parameters of purified biomolecules such as protein

tightly controlled experiments performed outside a living organism

allows variables to be isolated, but that doesn’t accurately reflect behaviors of complex bio systems so must be confirmed by vivo

Question 2

in vivo assays

Answer 2

conducted in whole living organisms with thousands of different molecules present

Question 3

Western blot

Answer 3

technique used to detect presence of specific proteins

1. load onto gel -> electrophoresis (moved by size)
2. proteins transferred to protein-binding membrane to become immobilized
3. any proteins not immobilized will become so with BSA or milk
4. membrane incubated with antibodies that specifically bind protein of interest
   - sometimes detected directly by markers or by addition of secondary antibody which binds to primary antibody
5. antibodies detected by fluorescence or chemoilluminescence

Question 4

isoelectric point

Answer 4

pH when the amino acids has a net charge of 0

Question 5

What proteins have the highest pI?

Answer 5

basic amino acids because the positive charge will need a higher pH to deprotonate the + side chains to become neutral

Question 6

What proteins have the lowest pI?

Answer 6

acidic amino acids because the negative charge requires a lower pH for neutralization

Question 7

What occurs during acetylation?

Answer 7

increases gene expression by freeing up histones

also neutralizes salts by neutralizing the amino acid’s positive charge

Question 8

circular dichroism

Answer 8

an absorption spectroscopy method based on the differential absorption of left and right circularly polarized light. Optically active chiral molecules will preferentially absorb one direction of the circularly polarized light.

can assess secondary structure in proteins

Question 9

gel elecrophoresis

Answer 9

separate based on size, charge, and shape

apply electric field with cathode and anode

larger molecules move slower so remain higher on gel

negative charge moves quicker toward anode than positive so will be further down gel

more aerodynamic shapes move quicker down gel

Question 10

SDS-PAGE

Answer 10

type of electrophoresis that adds SDS which will denature the protein and cause them to be separated solely based on size

adds something that makes all proteins the same negative charge and will all equally want to move toward the anode, but will stop in different locations based solely on molecular weight

heavier objects remain at the top, lighter objects found at the bottom

Question 11

reduced SDS-PAGE

Answer 11

type of electrophoresis that adds SDS and also a reducing agent so that disulfide bonds are broken

Question 12

Native-PAGE

Answer 12

does not reduce or denature proteins- UNALTERED

applies electric field to original protein - maintains original structure

separates based on charge and mass

ex: double stranded DNA is not torn apart

Question 13

Northern Blot

Answer 13

RNA

used after electrophoresis separates based on size

immobilized RNA with UV light
adds probe that is usually complementary strand with llabel to find specific RNA strand

Question 14

Southern Blot

Answer 14

DNA

used after electrophoresis separates based on size

immobilizing DNA with UV light
separates into single strand
adds probe that will locate specific DNA using label

Question 15

ELISA

Answer 15

enzyme-linked immunosorbent assay

used to see if proteins bind to specific antigens

Question 16

direct ELISA

Answer 16

1. antigen is immobilized
2. primary antibody is washed over
3. color change is reported by reporter gene if binding occurs

Question 17

indirect ELISA

Answer 17

1. antigen is immobilized
2. primary antibody wash
3. add secondary antibody that can recognize primary antibody
4. reporting of color change by reporter gene

Question 18

sandwich ELISA

Answer 18

see how much antigen is present

has primary and secondary antibody attach to the antigen

stronger color indicates a higher concentration of bound antibody reporters

Question 19

precise

Answer 19

how reliable something is

if something is precise, you will have similar results obtained for repeated trials

the scale saying you weight 120 lbs every time you get on it

Question 20

accurate

Answer 20

how correct the data represents real world value

this is assessed by comparison against gold standard

your phone saying it is 8:45am and comparing it to the world clock which says it is 8:45am

Question 21

calibration must be

Answer 21

compared against gold standard

Question 22

valid

Answer 22

both precise (very close in details) and accurate (giving correct information)

every time you get on the scale it says 120lbs. When you step on a scale that is calibrated corrected, it also says 120 lbs. So your scale is both precise and accurate so it is valid.

Question 23

affinity chromatography

Answer 23

separates based on highly specific noncovalent binding interactions

only the target protein will be bound and then everything else will be washed away.

Second wash, the target protein will be washed into a solution that we can observe

Question 24

cation exchange

Answer 24

columns are negatively charged and bind cations

so as the pH increases to become more basic (+) the more acidic peptides will elute sooner

Question 25

anion exchange chromatography

Answer 25

columns are positively charged and bind anions

will bind negative atoms so positively charges peptides will elute faster

Question 26

size exclusion chromatography

Answer 26

separates molecules by size

small molecules move slowly through size exclusion columns than do large molecules

OPPOSITE OF GEL ELECTROPHORESIS

Question 27

Why do D-amino acids have a longer half life than L-amino acids?

Answer 27

proteases cannot act on peptides made of D-amino acids because of the odd angles the side chain protrudes

therefore enzymes essentially work exclusively with L-amino acids and no not affect D-peptides

Question 28

stacking interactions

Answer 28

can stabilize secondary structures

occur between aromatic side chains - F, Y, P

Question 29

proline in secondary structures

Answer 29

is rigid and can adopt cis-configuration which is conducive to tight turns in beta sheets

Question 30

glycine in secondary structures

Answer 30

flexibility is favorable for beta turns

Question 31

why do hydrophobic portions aggregate

Answer 31

disruption of protein’s native conformation may force exposure of hydrophobic residues to the aqueous environment. The hydrophobic residues are unable to form hydrogen bonds with water, so when they are exposed to aqueous environment, water molecules will form rigid network to satisfy hydrogen bond requirements and this is thermodynamically unfavorable

that is why hydrophobic molecules aggregate to decrease total surface area exposed to water

Question 32

parallel beta sheets

Answer 32

parallel strands run in the same direction
N-terminal portion of one strand aligns with the N-terminal of the others

hydrogen bond pairs are slightly offset

lined by 360 degree loop

Question 33

antiparallel beta sheets

Answer 33

individual strands run in opposite directions from each other so N-terminal of one strand lines up with C-terminal of neighboring strand

hydrogen bond pairs directly align

linked by 180 degree beta sheets

Question 34

molecular chaperone proteins

Answer 34

help facilitate proper folding of other proteins

usually bind hydrophobic regions of nascent, misfolded, or aggregated proteins, preventing exposure of those regions to aqueous solvent

Question 35

negative transcription factor

Answer 35

downregulates transcription

Question 36

which amino acids are glucogenic?

Answer 36

leucine and lysine can be converted in to glucose through gluconeogenesis

glucogenic amino acid –> pyruvate –> oxaloacetate –> PEP

occurs through deamination

Question 37

deamination

Answer 37

alpha amino acid + alpha ketoglutarate –>glutamate + keto acid

ex: alanine + alpha ketoglutarate –> pyruvate + glutamate

taking off the amine group and adding oxygen

Question 38

irreversible inhibitors

Answer 38

react with nucleophilic side chains, forming covalent bonds to the enzyme

Question 39

preincubation with an inhibitor

Answer 39

provides more time for covalent linkages to occur and can increase the level of inhibition because more enzymes will be inactivated

Question 40

reversible inhibitors

Answer 40

bind target enzymes noncovalently

generally occur much faster, do not change with preincubation

Question 41

nucleophilic amino acids

Answer 41

R-groups contain atoms which donate electrons when deprotonated

serine, threonine, cytoseine, tyrosine, and lysine

Question 42

negative control

Answer 42

something is added where we know that no response will occur

ex: a study is looking at new types of fertilizer and how that will improve plant growth

negative control is adding no fertilizer. That way we can truly see if the treatment had an effect

other examples: placebo group, population that doesn’t receive any treatment

Question 43

positive control

Answer 43

something added that we know will have an effect and we expect results to occur

ex: a study is looking at how new fertilizer improves plant growth

positive control is using a fertilizer that is known to improve growth by 20%. That way we can see if the treatment is effective

Question 44

loading control

Answer 44

protein with high and ubiquitous expression

used to make sure that the protein has been loaded equally across all wells

should show up with all the wells having the same exact response

Question 45

Co-Immunoprecipitation

Answer 45

common technique used to identify an interaction between two proteins

Question 46

What type of SDS-PAGE is used for proteins?

Answer 46

proteins are smaller than DNA so loaded on a highly crosslinked polyacrylamide gel instead of agarose gel (which has larger pores)