Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Advanced Analytical Techniques > Immunoprecipitation > Flashcards

Immunoprecipitation Flashcards

1
Q

what is the principle of immunoprecipitation?

A
  • specific antibodies are used to bind to the antigen of interest
  • the antibodies are then bound to protein A/G beads
  • the protein beads can then be precipitated using centrifugation or magnetism
  • this allows the protein of interest to be isolated from a mixture of proteins
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

what are the different types of immunoprecipitations?

A
  • co-immunoprecipitation (Co-IP)
    • analyze protein-protein interactions
  • chromatin-immunoprecipitation (ChIP)
    • investigate regions of genome associated with a particular region of the genome
  • RNA-immunoprecipitation (RIP)
    • study the physical association between individual proteins and RNA molecules in vivo
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

what is co-immunoprecipitation?

A

this is a technique used to analyze protein-protein interactions, this is done by isolating one protein and investigating the proteins that can be found attached to it.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what are the general steps involved in co-immunoprecipitation?

A

Sample preparation
Pre-clearing
Antibody incubation
Precipitation of protein/protein complexes
Washing
Elution and analysis of precipitate

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

sample preparation what is it

A

mild detergents are used to lyse cells, the proteins are then placed on ice and protease inhibitors are used to prevent protein degradation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

pre-clearing what does it include

A

off-target ab/isotype control, as there can be proteins with similar properties to the target protein or proteins that will bind to antibodies non-specifically (this increases the accuracy/specificity of the immunoprecipitation.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

antibody incubation step explanation

A

the antibodies can be preloaded to beads or after the antigen-antibody interaction, the solution should be agitated to ensure complete binding

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

precipitation of protein/protein complexes explain step

A

centrifugation to cause pelleting or magnetism

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

washing - explain step

A

this is to remove impurities from the proteins

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

elution and analysis of precipitate - explain step

A
  • elution buffer is used to break electrostatic interaction between the target and antibody so that the protein can then be analysed
  • this can be:
    • SDS-PAGE
    • Western blotting
    • mass spectrometry
    • tandem mass spectrometry can also be used to analyse the AA sequence of the protein
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

elution and analysis of precipitate - explain step

A
  • elution buffer is used to break electrostatic interaction between the target and antibody so that the protein can then be analysed
  • this can be:
    • SDS-PAGE
    • Western blotting
    • mass spectrometry
    • tandem mass spectrometry can also be used to analyse the AA sequence of the protein
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

biological example of a co-immunoprecipitation experiment:

A

eIF4E-transporter protein, 4E-T:

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

eIF4E-transporter protein, 4E-T what is it

A
  • needed to know if 4E-T also bound to decapping factors; CNOT1, PATL1, LSM14 + DDX6
  • it was found that 4E-T interacts with 3 of the 4 mRNA decapping and decay machinery in a manner that is independent of its binding to elF4E
    pay attention to the western blotting seen on the right side of the image, might be required to read one and understand if it is a positive result ETC.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

what is chromatin immunoprecipitation?

A
  • it is a technique used to:
    • investigate regions of genome associated with a protein
    • identify specific proteins associated with a particular region of the genome
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

what are the problems with chromatin-immunoprecipitation?

A
  • it is hard to complete
  • it is prone to errors
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

what are chromatin immunoprecipitation applications?

A
  • DNA sequences occupied by specific protein targets
  • the binding sites and distribution of a particular protein, such as transcription factor, throughout the entire genome, under specified cellular conditions
  • gene transcription and RNA polymerase activity
  • complex DNA/protein interactions underlying disease phenotypes
  • modification to protein, such as histones, that many influence chromatin structure and gene expression
  • nucleosome architecture and regulation of chromosomal maintenance
17
Q

what are the general steps of chromatin immunoprecipitation?

A
  1. cross-link and harvest cells - fix proteins to DNA, using formaldehyde
  2. cell lysis + chromatin fragmentation - this is to break the chromatin into manageable sizes
  3. immunoprecipitation - this is when the chromatin is bound to an antibody
  4. wash, elution and cross-link reversal - this purifies the solution and then once pure unbinds the chromatin from the antibody
  5. DNA cleanup and analysis - this is with DNA purification methods, it can then be analysed with: PCR, qPCR, microarray or sequencing techniques
18
Q

antibodies - overview

A
  • they require highly epitope-specific Ab that recognises protein/residues of interest in their native chromatin states or possible cross-linked formation
  • some targets are more difficult to ChIP due to associations with other proteins/structures in vivo which can mask the epitopes
  • theoretically anything associated with chromatin can be ChiPed, but only if an antibody can be raised for it
  • commercially available antibodies are recommended
19
Q

ChIP - cross-linking stage:

A

Formaldehyde which works by cross linking amino groups between two amino acids

20
Q

ChIP - cell lysis + sonication of DNA:

A

cell lysis releases the chromatin from the cell and then sonication is used to break down the chromatin fragments; fragments are broken down to about 500bp size

21
Q

Purification steps:

A
22
Q

Analysis of ChIP DNA:

A

identification of DNA regions associated with the protein/modification of interest
PCR + qPCR - to look for target sequence
genome wide mapping of DNA binding proteins -DNA microarray + sequencing

23
Q

PCR + qPCR - to look for target sequence , what are the limitations?

A
  • use of primers bias towards the sequences of interest
  • makes the identification of unknown binding sites unlikely.
24
Q

what are the controls for ChIP:

A
  • negative controls:
    • input DNA:
      • a chromatin sample is processed parallel to the other samples but lacks the immunoprecipitation step
    • No Ab control:
      • a chromatin sample processed parallel to the other samples but immunoprecipitated without specific antibody
    • Isotype Ab control:
25
Q

% input versus fold enrichment: what is the main difference between the two?

A

fold enrichment doesn’t take into account the input only the noise

26
Q

ChIP-chip approach:

A
27
Q

ChIP-seq approach:

A
28
Q

what is RNA immunoprecipitation (RIP)?

A
  • it is a technique used to:
    • study the physical association between individual proteins and RNA molecules in vivo
29
Q

what are the general steps in RNA immunoprecipitation (RIP)?

A
  • it is a very similar process to that of ChIP
  • cross-linking can be applied but doesn’t have to
30
Q

immunoprecipitation and affinity chromatography combined:

A
31
Q

Systemic lupus Erythematosus (SLE): what is it

A

it is an autoimmune disease
- relapsing and remitting disease
- can be fatal as immune dysregulation and metabolic disturbances - accelerates cardiovascular disease
- characterised by a production of a range of autoantibodies - these target nuclear and cytoplasmic antigens

32
Q

What does SLE affect?

A
  • affects numerous systems:
    • renal
    • hematologic
    • musculoskeletal
    • pulmonary
    • cardiovascular
    • reproductive
    • neuropsychiatric
    • integumentary
33
Q

what do the autoantibodies target in 30-40% of SLE patients?

A
  • small nuclear ribonucleoprotein (snRNP) particles - a class of RNA-containing particles in the nucleus of eukaryotic cells involved in splicing to form mature mRNA.
  • snRNP’s; U1, U2, U4, U5, U6 form the spliceosome - this removes the introns from the precursor mRNA molecule and joins the exons to form the mature mRNA molecule.
  • all of the snRNP’s can be targeted by autoantibodies in the patients
34
Q

what do the autoantibodies target in 30-40% of SLE patients?

A
  • small nuclear ribonucleoprotein (snRNP) particles - a class of RNA-containing particles in the nucleus of eukaryotic cells involved in splicing to form mature mRNA.
  • snRNP’s; U1, U2, U4, U5, U6 form the spliceosome - this removes the introns from the precursor mRNA molecule and joins the exons to form the mature mRNA molecule.
  • all of the snRNP’s can be targeted by autoantibodies in the patients
35
Q

what are the snRNP’s made from?

A
  • they each consist of uridylate-rich small nuclear RNA (snRNA)
  • complexed with seven ‘sm’ proteins known as B, D1, D2, D3, E, F and G.
    • in addition to these U1 snRNP particles also contain other proteins; U1A, U1C, U1-70K
    • U2 snRNP particles also contain protein; U1A
36
Q

what does the autoantibodies towards the spliceosome cause?

A
  • there are abnormal mRNA transcripts as a result
  • this leads to abnormal proteins being produced
  • causes myriad of symptoms across a range of body systems
37
Q

Assay principle to identify spliceosome polypeptides targeted by autoantibodies:

A
  • five proteins (U1A, U1C, smD1, sm E1 + U1-70K) have been generated in vitro fused to beta galactosidase; pSV-B-galactosidase mammalian expression vector was used.
  • plasmids were then transfected into Cos-1 cells
  • fusion proteins were then present in the Cos-1 lysates
  • the fusion proteins were then mixed with patients sera and protein A/G beads - the beads are able to bind all subclasses of IgG, IgA, IgM + IgE.
  • after incubation of the serum with the beads and fusion proteins, the sample will be centrifuged to pellet the A/G beads which will be associated with the patients antibodies.
  • if there has been no binding of the antigen then it will be washed away in the washing steps.
  • detection step uses the B-gal activity of the fusion protein to detect whether there is interaction between the protein and an antibody in the patients serum - the B-gal activity is measured using the substrate O-nitrophenyl-B-D-galactopyranoside (ONPG) (which is cleaved by the enzyme) —-—> B-D-galactose and o-nitrophenol (absorbs light at the wavelength 420nm).
38
Q

how could the method be made quantitative?

A
  • a standard curve could be drawn from known antibody concentrations absorbance
  • the absorbance of unknown samples could then be used to determine the concentration of the antibodies in the sample
39
Q

how can positive results be determined?

A

the wells with a higher absorbance are those that are positive

Advanced Analytical Techniques (8 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions