L3 - More Biochem Techniques Flashcards

1
Q

how do you monitor protein overexpression?

A

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
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2
Q

cell lysis

A

extract all proteins in cell using a buffer

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3
Q

SDS-PAGE

A

separate proteins according to MW

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4
Q

what kind of proteins are separated in SDS-PAGE?

A

denatured proteins

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5
Q

how are proteins denatured in SDS-PAGE?

A

1) SDS detergent

2) heating

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6
Q

what kind of gel used in SDS?

A

polyacrylamide gel

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7
Q

when do you use loose gels in SDS-PAGE?

A

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

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8
Q

when do you use tight gels in SDS-PAGE?

A

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

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9
Q

immunoblotting

A

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

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10
Q

what kind of signal is seen in WB?

A

chemiluminescence

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11
Q

what kind of gels are used in immunoblotting?

A

1) PVDF

2) nitrocellulose

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12
Q

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

A

protein ladder (molecular weight markers

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13
Q

what are the applications of WB?

A

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

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14
Q

loading control

A

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

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15
Q

when do you use loading controls?

A

when comparing different cell types, conditions, etc.

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16
Q

what are common proteins used in loading control?

A

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

17
Q

how is protein’s activity usually controlled?

A

its phosphorylation status

18
Q

how can a protein’s phosphorylation status be assessed?

A

1) WB

2) phospho-specific Abs

19
Q

ELISA

A

enzyme-linked immunosorbent assay

20
Q

how is ELISA used?

A

detect small, soluble molecules

21
Q

describe ELISA process

A

1) ELISA sandwich

2) measure absorbance

22
Q

why is ELISA so good?

A

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

23
Q

how is immunoprecipitation used?

A

identify protein X’s interacting partners in cells

24
Q

describe immunoprecipitation process

A

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

25
what is the limitation of immunoprecipitation?
does not indicate whether interactions are direct
26
for cells with fusion proteins, what kind of Ab can you use in IP?
Ab against its tag or protein itself