How are proteins studied?

Question 1

4 methods to detect proteins inside cells

Answer 1

1. immunofluorescence
2. fluorescent conjugates
3. fluorescent fusion proteins
4. tagged fusion proteins

Question 2

immunofluorescence

Answer 2

fluorescein is covalently attached to an antibody which binds to a specific protein

Question 3

to types of immunofluorescence

Answer 3

direct or indirect

Question 4

fluorescent conjugates

Answer 4

attach a fluorescent molecule to another molecule with an affinity for a specific protein

Question 5

example of a fluorescent conjugate

Answer 5

phalloidin has a natural affinity for actin filaments

Question 6

most common fluorescent fusion protein and its source

Answer 6

green fusion protein (GFP) from jellyfish

Question 7

where are fusion genes made?

Answer 7

in vitro

Question 8

example of a fluorescent fusion protein

Answer 8

in yeast cells with POT1::GFP fusion gene, proteins will show up in peroxisomes

Question 9

tagged fusion proteins

Answer 9

if there are no antibodies available for a specific protein, fusion genes can add on an HA tag sequence

Question 10

HA tag sequence

Answer 10

region of the fusion protein that antibodies can bind to

Question 11

3 methods to isolate proteins

Answer 11

1. total proteins
2. immunoprecipitation
3. co-immunoprecipitation (Co-IP)

Question 12

total proteins

Answer 12

proteins can be isolated from tissues, cells, or organelles

Question 13

immunoprecipitation

Answer 13

specific proteins can be purified with antibody columns

Question 14

immunoprecipitation procedure

Answer 14

1. equilibrate column
2. apply crude proteins to column
3. add wash buffer
4. add elution buffer

Question 15

co-immunoprecipitation

Answer 15

used to determine if 2 proteins are normally attached in cells

Question 16

2 methods to detect proteins outside cells

Answer 16

1. SDS-polyacrylamide gel electrophoresis (SDS-PAGE)

2. Western blot hybridization (western blotting)

Question 17

purpose of SDS-PAGE

Answer 17

to quantify all proteins present in a sample

Question 18

4 step procedure of SDS-PAGE

Answer 18

1. proteins coated in SDS to gain a net negative charge
2. proteins loaded into polyacrylamide gel
3. power is applied and proteins migrate to the positive electrode
4. gel is soaked in a protein stain

Question 19

polyacrylamide gel

Answer 19

tangle of fibers to provide an obstacle in front of proteins

Question 20

2 commonly used stains for SDS-PAGE

Answer 20

coomassie brilliant blue or silver nitrate

Question 21

which step from SDS-PAGE does western blotting replace

Answer 21

step 4 (soaking in a protein stain)

Question 22

Western blot procedure (cont’d from after step 3 in SDS-PAGE)

Answer 22

1. remove gel and transfer proteins to a membrane
2. incubate membrane with antibodies (soak)
3. wash
4. incubate the membrane with detection reagents

Question 23

which 2 procedures are commonly used together?

Answer 23

Co-IP and Western blot

Question 24

3 methods to eliminate specific proteins inside cells

Answer 24

1. gene mutations
2. morpholinos
3. antibody depletion

Question 25

gene mutations

Answer 25

cells with a non-functioning gene can't make a corresponding protein

Question 26

morpholinos

Answer 26

synthetic mRNA-like molecules that bind to target mRNAs preventing protein synthesis

Question 27

antibody depletion

Answer 27

antibodies bind to proteins within a cell and stop them from functioning