3 Flashcards

1
Q

Method of centrifugation?

A
  1. Fractionation- e.g. ground in a blender, osmotic shock, ultra-sonication (sound waves agitate) (Detergents?) to break the PM’s and free the organelles while keeping them in tact largely.
  2. This is called the cell Homogenate, with cell fractions.
  3. Preparative ultracentrifuge rotates the broken cells at high speed to separate the contents by size and density.
  4. High/ Low? density organelles rotate less so settle at the bottom as a pellet e.g. mitochondria, then at a slightly higher speed, ribosomes.
  5. These centrifugations may be repeated several times. E.g. initially at low speed 5G, and then this supernatant spun again at high speed 100,000G.
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2
Q

Velocity Sedimentation?

A

When centrifuged the components move as a series of distinct bands each at a different rate. To stop mixing (when denser towards the bottom) a density gradient is made by adding a shallow gradient of sucrose (5 to 20% going down). When centrifuged now distinct bands of sediment are created which can be separated, by collecting as drops from the bottom after puncturing the tube.

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3
Q

Equilibrium Sedimentation?

A

Ultracentrifugation.
Allows components of the cell to be separated by Buoyant density, independent of size or shape. The bands are created by a deep sucrose gradient (20-70%), and when the density of the organelles is the same as the sucrose they stay in position, with those at the bottom of the highest buoyancy density.

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4
Q

Ion exchange chromatography?

A

Ion Exchange: separates by Charge. Generally, proteins are slightly negative, so they are attracted to the positive beads, and any cations are lost. After, increasing conc of salt are used to compete for the ionic interaction between protein and bead, separating them fraction by fraction depending on charge. The positive beads are either Cation exchange Carboxy-Methyl beads (CM) or anion exchange DEAE.

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5
Q

3 types of chromatography?

A

Ion exchange: separates by charge, negative proteins stick to positive bead
Gel filtration: sepatates by size as the small molecules go into the beads and are retarded
Affinity chromatography: separates by binding, beads with covalent bound antibody to

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6
Q

Gel filtration chromatography?

A

Porous beads are used, so the small molecules are retarded as go through the beads, whereas larger move through and excluded faster, so separated by size.

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7
Q

Affinity chromatography?

A

Affinity Chromatography: Binding. If want to separate a known enzyme for example, can make a bead with covalently bound substrate to that enzyme, so it binds are is not excluded. Or DNA oligos to a specific DNA sequence. High salt or acid is then used to separate this bound protein. This creates a very pure fraction even single pass.

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8
Q

What is Gel electrophoresis for?

A

Separating proteins by molecular weight for analysis- can tell which proteins are present when compare to known.

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9
Q

Method of 2D Gel electrophoresis?

A

2D Gel Electrophoresis:
Used to achieve greater purity for complex samples. It uses Isoelectric focussing in the first dimension, separating both by charge, and SDS PAGE separating by size in the second dimension. At certain PH proteins lose their charge (denatured) so stop running at their Isoelectric point.

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10
Q

SDS-PAGE gel electrophoresis?

A

SDS-PAGE:

  1. SDS- detergent molecule which denatures and unwinds the protein into sticks exposing the hydrophobic molecules hidden in the centre.
  2. B-Mercaptoethanol can be used to break disulphide bonds between polypeptide subunits. Boiled for two minutes in these two.
  3. Load proteins into well and as negative run through the Polyacrylamide gel electrophoresis (PAGE) to the Anode.
  4. Protein migration is proportional to the molecular weight, so separates out proteins.
  5. This can be used to analyse how pure a protein sample is.
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11
Q

Difference between gel electrophoresis 2D or SDS?

A

2D separates by charge and molecular weight, whereas SDS separatesd by molecular weight only

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12
Q

Immuno/western blotting used for?

A

Visualising samples e.g. after electrophoresis.

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13
Q

Method of western blotting?

A
  1. Transfer the proteins onto a sheet of nitrocellulose paper by driving the proteins out of the gel using a strong electric current.
  2. Complimentary primary antibodies are made to the protein.
  3. Wash off excess.
  4. A second antibody then hybridises with this primary, which are tagged with fluorescent dyes or radioactive isotopes, so can be visualised.
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14
Q

Why is a secondary antibody used to the primary antibody?

A

Because it is more economical, e.g. can be amplified, and it is more efficient, for example the secondary antibody can be bought that are tagged and these will bind to other antibodies such as the primary. This means you dont need to tag the specific antibodies.

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15
Q

What are the origins of the antibodies used in immunoblotting/western?

A

The primary is often mouse or rabbit. The secondary anti-rabbit or anti-mouse are often donkey, and joined to horseradish peroxidase.

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16
Q

How are reporter lines done?

A

The reporter gene e.g. GFP is electroporated into the gene under the same promoter, so whenever the gene is expressed so is this, which can be visuallised as fluorescence.

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17
Q

How does immunofluorescence work?

A

Immunofluorescence (IF) is a detection method, during which antibody binding to an antigen is visualized using a fluorophore attatched to a secondary antibody, which attatches to the primary to the antigen of interest.

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18
Q

Methods for purifying proteins?

A
  • Centrifugation- separates by size and density
  • Chromotography:
    Ion exchange: charge
    Affinity: Binding
    Gel filtration: Size
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19
Q

Purpose of gel electrophoresis?

A

Can tell how pure a sample is e.g. if only one band =pure.
Can use to see protein binding if more than one band or units e.g. Light chain and heavy
Can help identify proteins/Strands by molecular weight.

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20
Q

What is mass spectrometry used for?

A

Used to identify proteins by comparing to known samples.
Also, v sensitive so can show if protein has anything bound or phosphorylated.
Estimate number of AA in a sample.

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21
Q

Method for mass spec?

A
  1. After Protein isolation, Trypsin may be used to cleave the protein into single peptides.
  2. Mass spectrometry exploits the difference in ions mass/charge ratio (m/z).
  3. The sample is turned into a gas in the ion source.
  4. The sample is separated by an electric field by this ratio in the mass analyser.
  5. The detector creates the peaks which can then be compared to known samples to identify.
22
Q

Experimental techniques for visualising proteins? (3)

A

Western blotting- Plymouth= proteins e.g. after electrophoresis
Immunofluorescence

23
Q

techniques for protein interractions? (8)

A
Pull-down method
Immunoprecipitation
Affinity chromotography
GST affinity pull down
DNA footprinting
EMSA
Gel electrophoresis
Yeast-2-hybrid screen
24
Q

Experiments to show protein interraction strength?

A

DNA Chromatography
A radioactivity based binding
Surface plasmon resonance

25
Q

What is immunoprecipitation?

A

Immunoprecipitation is an alternative to affinity binding, where specific antibodies are attached to agarose beads. This is not done in a column, but just added to a test tube. The beads are then collected by low speed centrifugation.
Can be used to show protein-protein interractions also.

26
Q

What are reporter lines used for?

A

Visualising where mRNAs of a gene are located and when.

27
Q

Method of reporter lines?

A

Cis regulatory DNA sequences located near the coding sequence control gene transcription. If place a reporter gene under their control and introduce these recombinant DNA molecules into the cells, these will be expressed when and where the protein of interest is. These reporter genes often code a fluorescent protein e.g. GFP so can be visualised.

28
Q

How does FRET work? Use?

A

Relies on paired fluorescence in order to get green out, so can use this to determine whether proteins interact. As the molecules are excited by one wavelength and emit another. This not only shows protein interaction but also the location of in situ.

29
Q

PCR method?

A
  1. Separate two strands by heating to 95 degrees.
  2. Annealing of DNA oligonucleotide primers at much cooler temperatures (known sequence complimentary to at opposite ends of two strands)
  3. Elongation of DNA using oligonucleotides added using a thermo-stable DNA polymerase e.g. Taq polymerase at 72degrees.
  4. Repeated again.
30
Q

3 types of centrifugation?

A

Differential: Keep separating the supernatant and pellet by density, with increasing speeds
Velocity: By density, but then use a sucrose 5-20%- more dense bottom
Equilibrium: Bouyancy density separates 20-70% sucrose- stays when at same density

31
Q

How does horseradish peroxidase work?

A

When the antibody binds to the antigen, there is an enzyme reaction that happens- oxidation, and light or colour is given off, e.g. Enzyme-Linked Immunoabsorbant Assay (ELISA) for Herceptin.

32
Q

What is mass spectrometry used for? (3)

A
  • After protein has been isolated, mass spec can be used to identify proteins by comparing their mass/charge ratio to known samples.
  • It’s very sensitive so can be used to show if proteins are labelled, as these will give a specific number higher e.g. if phosphorylated.
  • Because its so precise can also be used to estimate the amino acids in.
33
Q

Method of mass spec?

A
  1. After Protein isolation, Trypsin may be used to cleave the protein into single peptides.
  2. Mass spectrometry exploits the difference in ions mass/charge ratio (m/z).
  3. The sample is turned into a gas in the ion source.
  4. The sample is separated by an electric field by this ratio in the mass analyser.
  5. The detector creates the peaks which can then be compared to known samples to identify.
34
Q

Method of immunoprecipitation?

A
  1. Tag the protein of interest with peptide DYKDDDDK tag.
  2. Add other proteins that may bind to the protein of interest.
  3. Use a specific Antibody to the tag.
  4. Add Protein A coated beads complimentary to the antibody bind strongly.
  5. Centrifuge to recover the complex.
  6. Identify by mass spectrometry or Western Immunoblotting.
35
Q

Affinity chromatography used for?

A

Protein purification

Protein-interractions (add prey to bait protein that will stick to the bead and then run on SDS PAGE to separate after)

36
Q

Immunoprecipitation used for?

A

Protein purification and protein to protein interractions

37
Q

Method of GST affinity pull down?

A

Version of affinity chromatography.

  1. GST expressed on the protein of interest using recombinant techniques.
  2. Bind to the the bead via GST to Glutathione.
  3. Prey interracting proteins will bind to the POI so not be eluted.
  4. Eluted by inserting Glutathione solution which will compete with the beads and gradually elute the complexes.
  5. separation by Resulting mixtures separated by SDS-PAGE and western blotted with antibodies for binding partners.
  6. If binding partners unknown, identify by mass spectrometry.
38
Q

Method of DNA footprinting?

A

Radioactively label one end of the DNA (32P) =probe.
When there is no protein bound the DNase randomly cleaves the DNA sequences, creating many different length strands. However, when there is a protein interacting, this protects this area of DNA, so it cannot be cleaved at this length.
Heat the sample to destroy DNase and release the interacting protein.
When run on SDS-PAGE electrophoresis this molecular weight is left blank (DNA Footprint) where the protein is bound.

39
Q

DNA footprinting used for?

A

Showing protein-protein interractions and location, not what the protein is, but other methods could be used to find that out.

40
Q

EMSA method?

A

Radioactively label one end of the DNA, and mix this probe with protein of interest. Run this on gel electrophoresis and if the protein doesn’t interact then it will move further faster, however if it is then it will move more slowly, and this can be seen and shows interaction. Do not heat up the sample, unlike above, so the protein isn’t released.

41
Q

EMSA used for? Name?

A

Electrophoretic Mobility Shift Assay.

Testing whether different proteins interract to a POI

42
Q

How is DNA electrophoresis different to SDS PAGE?

A

For DNA instead of proteins.

Can show protein interractions with DNA as will increase the molecular weight.

43
Q

Yeast two-hybrid screen method?

A
  1. A transcription factor is split into two parts; the DNA binding domain and the transcriptional activation domain.
  2. The DNA binding domain is attatched to the POI bait.
  3. The activation domain is attatched to the prey protein.
  4. These are introduced into a yeast cell in a plasmid.If the bait and prey do interract these domains will be close enough to allow for transcription of the gene.
  5. This could be visuallised/ shwon by this gene being a GFP or antibiotic resistance gene.
44
Q

Yeast-two hybrid screen used for?

A

Showing protein/protein interractions.

45
Q

DNA chromotography method?

A

After affinity chromatography, start with a low salt strength and see which proteins are eluted. Gradually as increase the salt concentration other proteins will be eluted. The strongest interactions will require the strongest salt solution to dislodge them.

46
Q

DNA chromatography use?

A

work out protein strengths of interraction

47
Q

Radioactivity based binding method?

A

Plot the amount of protein A versus bound
(AB complex) you can generate a binding curve.
From such curve you can derive the max binding
stoichiometry – how many A bind to B and the
association constant, i.e. how tightly A binds to B, which
is equivalent to the affinity of A to B. The stronger they interact the steeper the curve

48
Q

Radioacitivty based binding use?

A

Protein interraction strength

49
Q

Surface plasmon resonance (Biacore) method?

A

The binding of prey molecules to bait chnages the refractive index, altering the resonance angle for plasmon induction.
This can be detected by a detector.
The number associated before vs dissociates after buffer solution added creates a curve.
The stronger the proteins interract the steeper the curve.

50
Q

Methods of showing protein interractoin strength?

A

Surface plasmon resonance (Biacore)
Radioactivity based binding
DNA chromatography