L3 - More Biochem Techniques

Question 1

how do you monitor protein overexpression?

Answer 1

couple DNA for protein of interest to DNA for a tag protein

• transfect the DNA into cells
• fusion protein produced by cells
• can be visualized by fluorescence microscopy
• tagged proteins can also be recovered w biochem techniques

Question 2

cell lysis

Answer 2

extract all proteins in cell using a buffer

Question 3

SDS-PAGE

Answer 3

separate proteins according to MW

Question 4

what kind of proteins are separated in SDS-PAGE?

Answer 4

denatured proteins

Question 5

how are proteins denatured in SDS-PAGE?

Answer 5

1) SDS detergent

2) heating

Question 6

what kind of gel used in SDS?

Answer 6

polyacrylamide gel

Question 7

when do you use loose gels in SDS-PAGE?

Answer 7

allow heavy proteins through (don’t discriminate light proteins well)

Question 8

when do you use tight gels in SDS-PAGE?

Answer 8

allow light proteins through (don’t discriminate heavy proteins well)

Question 9

immunoblotting

Answer 9

separated proteins are transferred to a membrane and incubated for an Ab with protein of interest, and then conjugated with a secondary Ab that gives a signal

Question 10

what kind of signal is seen in WB?

Answer 10

chemiluminescence

Question 11

what kind of gels are used in immunoblotting?

Answer 11

1) PVDF

2) nitrocellulose

Question 12

what is important to use in all immunoblotting experiments/WB?

Answer 12

protein ladder (molecular weight markers

Question 13

what are the applications of WB?

Answer 13

1) check expression of protein
2) compare proteins of interest between cell types
3) assess cell signalling pathways under different conditions

Question 14

loading control

Answer 14

confirm that equal amount of total protein was loaded onto each lane

Question 15

when do you use loading controls?

Answer 15

when comparing different cell types, conditions, etc.

Question 16

what are common proteins used in loading control?

Answer 16

1) GADPH
2) actin
3) tubulin
(basically most housekeeping proteins)

Question 17

how is protein’s activity usually controlled?

Answer 17

its phosphorylation status

Question 18

how can a protein’s phosphorylation status be assessed?

Answer 18

1) WB

2) phospho-specific Abs

Question 19

ELISA

Answer 19

enzyme-linked immunosorbent assay

Question 20

how is ELISA used?

Answer 20

detect small, soluble molecules

Question 21

describe ELISA process

Answer 21

1) ELISA sandwich

2) measure absorbance

Question 22

why is ELISA so good?

Answer 22

1) sensitive
2) quantitative
3) precise
4) high-throughput

Question 23

how is immunoprecipitation used?

Answer 23

identify protein X’s interacting partners in cells

Question 24

describe immunoprecipitation process

Answer 24

1) cell lysis
2) incubate cell lysate with bead-bound Ab against protein X
3) spin down beads (and protein X and its partners)
4) extract proteins from beads (heat)
5) separate extracted proteins via SDS-PAGE
6) blot associated proteins with Abs against suspected proteins

Question 25

what is the limitation of immunoprecipitation?

Answer 25

does not indicate whether interactions are direct

Question 26

for cells with fusion proteins, what kind of Ab can you use in IP?

Answer 26

Ab against its tag or protein itself