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Molecular Biology > Lec 3-4 > Flashcards

Lec 3-4 Flashcards

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1
Q

Why do we study protein function?

A
  • knockdown or gene knockout
  • transfection/ transformation overexpression studies
  • protein localization w/i cells
  • determine protein structure
  • in vitro function
  • identify interacting proteins
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2
Q

describe protein separation by SDS polyacrylamide gel electrophoresis

A
  1. proteins are boiled in strong SDS detergent to denature into the primary structure and make them negatively charged
  2. run the sample in gel, electrical current forces proteins to run downwards to a positive pole, separated by charge
  3. add coomassie blue to stain
  4. to detect specific proteins, can do western blotting if you have antibodies against the protein of interest, or if unknown, the protein can be identified by mass spectroscopy
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3
Q

what is the old way to purify proteins?

A

cell fractionation combined with vitro assay

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4
Q

what is immunoprecipitation?

A

a rapid form of affinity chromatography purification method

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5
Q

describe the steps of immunoprecipitation

A
  1. grind up cells in extraction buffer then centrifuge to obtain cell extract
  2. add an antibody against a specific protein, needs to bind at a high affinity
  3. add agarose beads coupled to a specific protein, then add detergent to elute everything else
  4. centrifuge and keep the pellet that contains beads-antibody-specific protein
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6
Q

how to purify protein without antibodies against it

A

clone the gene and express it in e.coli, can make lots of copies (overexpression)

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7
Q

how to obtain purified protein after expressing in e.coli

A

clone the gene into a vector with an epitope tag or affinity tag, express the fusion protein in bacteria or in vitro, then use IP to purify

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8
Q

what is an epitope tag?

A

a small protein sequence (10 a.a. long) that’s recognized by commercially available antibodies ex. HA tag

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9
Q

what is an affinity tag?

A

larger than an epitope tag, it’s a protein that can be easily purified based on its affinity for a specific substrate e.g. the GST tag has a specific binding affinity for glutathione

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10
Q

why do we express the tagged form of protein in bacteria and then purify it?

A
  • use in vitro assays
  • test for direct interactions b/w 2 purified proteins (GST pulldown)
  • determine protein structure
  • antibody production (study protein localization and perform co-IP to identify interacting proteins)
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11
Q

what is a fusion protein?

A

a protein fused with an epitope tag or affinity tag

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12
Q

how to make a fusion protein?

A
  1. use cDNA to amplify your gene by PCR - PCR primers also carry restriction site sequences for cloning
  2. clone your gene into a vector that already has an epitope tag or affinity tag in it (or for epitope tags, one of the primers contains an epitope sequence)
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13
Q

how to use PCR to insert an epitope tag?

A

PCR primers have restriction enzyme sites at ends that can clone the PCR product into a plasmid vector and 1 of the primers also has a sequence for an epitope tag ex. EcoR1 has HA

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14
Q

how do you make a fusion protein with a plasmid that already has an epitope tag?

A

clone into a GST (glutathione S-transferase) vector to express GST fusion protein

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15
Q

describe GST affinity purification

A
  1. clone gene of interest into GST vector, now express GST-protein x (gene of interest)
  2. bind GST-protein x to GSH (glutathione) beads, then mix and incubate
  3. wash beads, elute with GSH
  4. run eluate (exiting mixture) on protein gel
  5. now have purified GST-protein x
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16
Q

what is a GST pulldown?

A

tests for in vitro interaction between 2 known and cloned proteins
1. GST-protein x
2. HA-protein Y

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17
Q

what are the steps of a GST pulldown?

A
  1. bind GST-protein x to GSH beads
  2. incubate with HA-protein y
  3. wash beads, eluate with GSH
  4. run eluate on protein gel
    if protein y can bind to protein x, it will stick to the beads so if we wash them multiple times then it should stay attached
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18
Q

describe protein expression in animal cells

A
  • can express tagged form of protein in cultured cells or in whole organism
    i) vector contains a strong inducible promoter for animal cell expression
    ii) transfect into an animal to get transgenic animal
    iii) like bacterial expression, the gene is cloned with an epitope tag or affinity tag or with GFP
    iv) purify protein using epitope tag or affinity tag
    v) directly study protein localization
    vi) directly perform co-IP to identify interacting proteins
19
Q

describe co-IP

A
  1. grind up cells in mild extraction buffer to preserve protein-protein interactions, centrifuge to obtain cell extract
  2. add an antibody against the specific protein
  3. add beads coupled to the specific protein
  4. centrifuge pellet which contains beads-antibody-specific protein
  5. known associated proteins can be identified by western blotting and unknown associated proteins can be identified by mass spec
20
Q

what is mass spectrometry

A

a highly sensitive method for identifying unknown proteins, after co-IP the eluate is run on the SDS page, then stained with coomassie, and the prominent bands are cut out THEN the proteins are broken into peptides and ionized which determines the mass/charge ratio and software determine what combo of a.a. can give that mass/charge ratio and then you can search protein databases for predicted proteins with those a.a.

21
Q

what can you use to test interactions between 2 known proteins?

A
  1. GST pulldown (can they interact)
  2. CoIP, then Western (do they interact)
22
Q

what can you use to identify unknown interacting proteins?

A

CoIP, then mass spec (what interacts)

23
Q

what does BLASTp help identify?

A

related proteins in the same or other species (identifies homologues, paralgues)

24
Q

what does prosite help identify?

A

putative domains based on sequence similarity to characterized domains (possible functions, indicate relationship to known gene families)

25
Q

what does x-ray diffraction help determine

A

protein structure

26
Q

describe x ray crystallography

A
  1. start with the purified protein expressed in e.coli
  2. protein is dehydrated, makes crystals
  3. crystals are the source for x ray diffraction
  4. determines protein’s crystal structure
27
Q

what is NMR

A

nuclear magnetic resonance, it doesn’t require crystalization but is not as precise as crystallography and doesn’t work for large proteins

28
Q

what is immunofluorescence used for

A

detect specific proteins in cells

29
Q

how to generate antibodies

A
  1. purified protein is obtained from bacterial expression from in vitro translation OR from in vitro synthesis of a short peptide, corresponding to part of the protein
  2. protein is injected into an animal
  3. get serum after several weeks, which contains antibodies against multiple epitopes on protein and non-specific antibodies (fusion tag specific antibodies)
  4. the protein of interest is purified by affinity purification
30
Q

how are monoclonal antibodies generated?

A

mostly come from mice, the spleen is removed after immunization, and beta cells that produce the antibody are harvested, these fuse with myeloma cells to become hybridoma cells that can divide indefinitely and produce a single antibody, from the hybridoma cell line just 1 antibody specific to the injected protein is identified and the supernatant from that cell line contains pure mono-specific antibodies and the cell-line can live forever

31
Q

how can antibodies be used to detect specific molecules?

A

for immunofluorescence:
1. process cells for microscope slide, has immobilized antigen A
2. incubate slide with a primary antibody (rabbit antibody directed against antigen A)
3. a secondary antibody is marker-coupled and directed against rabbit antibodies, it attaches to the base of the primary antibody
4. now fluoresces

32
Q

how can antibodies be used to detect protein interactions?

A
  1. process cells for microscope slide, has immobilized antigen A and antigen B
  2. incubate slide with rabbit antibody directed against antigen A and mouse antibody directed against antigen B
  3. then marker-coupled goat anti-rabbit and marker-coupled goat anti-mouse attach
  4. now can see where protein is relative to other proteins
33
Q

what is the alternative to antibodies?

A

express as a fusion protein in vivo, then use epitope tag for western blotting and IF or GFP tag for live imaging (localization of protein)

34
Q

how does live imaging using GFP (green fluorescent protein) work

A
  1. clone gene into a plasmid that has GFP sequence and strong promoter for animal cells
  2. transform into animal cells
  3. image living cell with fluorescent microscope
35
Q

bacterial vs. animal cell expression

A

bacterial: purification of larger amounts, purer, better for crystal structure determination, antibody production, and in vitro studies
animal: directly study protein localization, protein processed properly

36
Q

describe gene knockdown by antisense oligo

A

the injection of RNA that’s complementary to a specific mRNA will bind and prevent the translation of the mRNA, therefore knocking down the gene

37
Q

what is expected from the sense control

A

it will not interfere with translation as it has the same sequence as mRNA so it won’t have any effect

38
Q

what is expected by combining the sense and antisense strands

A

they would reduce the effect of the antisense oligo as they would bind, but it resulted in over 100 times stronger knockdowns than antisense alone which led to the discovery of interfering RNA (translational repression or RNA degredation)

39
Q

describe RNA interference

A
  1. processing: dicer complex cleaves dsRNA into 21-23nt fragments called siRNA
  2. RISC cleaves 1 strand of siRNA, now passenger strand
  3. RISC mediates RNAi activity by binding guide strand to mRNA and mRNA cleaved for translational repression
40
Q

how can RNAi protect against RNA viruses

A

viruses and retrotransposons produce shRNA which allows dicer to process it into siRNA, then RISC can target the viral or transposon RNA

41
Q

what are the 2 parts of the CRISPR locus

A
  1. repeat sequences - regularly interspersed, conserved in bacteria
  2. spacer sequences - random DNA in between
42
Q

how does CRISPR/Cas9 work

A
  1. incoming viral DNA is recognized by Cas9 due to PAM sequence
  2. Cas1/2 endonuclease cleaves DNA at 5’ PAM sequence
  3. spacers get incorporated into genome at CRISPR locus
  4. CRISPR locus transcribed, Cas proteins process this pre-crRNA to crRNA and associates with tracrRNA and Cas9
  5. now guide RNA (crRNA + tracrRNA) deviates immune response next time virus infects but only recognizes viral DNA if it has complementary sequence to crRNA + 3’ PAM sequence
43
Q

how does CRISPR/Cas9 protect againt autoimmunity

A
  1. bacterial genome round, therefore can recognize linear DNA ends
  2. CRISPR locus doesn’t have PAM sequence on spacer sequence, therefore doesn’t recognize it
44
Q

how does gene knockout work by CRISPR/Cas9

A
  1. identify PAM sequence in gene of interest, design synthetic guide RNA that’s 20nt sequence upstream of PAM sequence + crisper repeat + tracrRNA
  2. introduce Cas9 into gene cell by transformation
  3. introduce guide RNA into cells which leads to knockout of specific gene because Cas9 mediates a ds break in the DNA and NHEJ repairs it but creates small deletion leading to RF shift and knockout

Molecular Biology (3 decks)

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