Protein Analysis Flashcards Preview

BMS 242 - Advanced Cell and Molecular > Protein Analysis > Flashcards

Flashcards in Protein Analysis Deck (43)
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1
Q

What are biochemical approaches to protein analysis?

A

Purify protein from a MIXTURE of proteins
and
Identify that protein

2
Q

Why do we need to purify proteins?

A

Cells contain ~20,000-30,000 proteins so need to isolate protein of interest

Can run tests on this purified protein to address the structure and function of the protein

3
Q

Why would we want to identify proteins?

A

Protein may have a specific activity:

  • Antibiotic activity
  • Fluorescent protein
  • May want to see WHY it has a certain activity - in order to identify similar proteins which may have the same activity
  • May want to isolate the activity and use it as a treatment
4
Q

What is proteomics?

A

Analysis of the complete protein content in a living system (PROTEOSOME)

INCLUDING ALL post-translationally modified proteins and alternatively spliced variants

5
Q

Why would you want to carry out proteomics?

A

To compare the proteins present in one cell to the proteins present in another cell

6
Q

What does typical protein purification involve?

A

1) Tissue homogenisation
2) Separation of the released material from unbroken material by centrifugation:
- Ion-exchange
- Gel filtration
- Affinity
3) Several column chromatography steps
4) Confirmation of protein purity after each purification step

7
Q

What can be used to homogenate tissue?

A

Sonication
Blending
Pestle and mortar

8
Q

Describe column chromatography

A
  • The column is packed with a matrix (many little beads)

- The beads have different characteristics (used to separate the proteins)

9
Q

In column chromatography, what can the proteins be separated by?

A

Size
Affinity
Ion exchange (separated out due to charge)

10
Q

What is column chromatography used for?

A

To separate out proteins, in order to concentrate the protein of interest - can study structure and function

11
Q

How can protein purity be tested? (4 ways)

A
  • Electrophoresis
  • Western immunoblotting
  • Mass-spectrometry
  • Protein specific assay
12
Q

What is the process of DIFFERENTIAL centrifugation?

A

1) Spin cell homogenate at LOW SPEED
- Increase G force on solution
- Organelles settle to the bottom
- Forms a pellet and a supernatant

2) Spin at MEDIUM SPEED
3) Spin at HIGH SPEED
4) Spin at VERY HIGH SPEED

13
Q

In differential centrifugation, what is contained in pellet 1?

A
  • Whole cells
  • Cytoskeleton
  • Nuclei
14
Q

In differential centrifugation, what is contained in pellet 2?

A
  • Mitochondria
  • Lysosomes
  • Peroxisomes
15
Q

In differential centrifugation, what is contained in pellet 3?

A
  • Microsomes

- Small vesicles

16
Q

In differential centrifugation, what is contained in pellet 4?

A
  • Ribosomes
  • Viruses
  • Large macromolecules
17
Q

In differential centrifugation, what is contained in the supernatant after spinning the homogenate at very high speed?

A

Pure cytosol

Highly soluble components

18
Q

After running DIFFERENTIAL cell centrifugation, what are you left with?

What is the next step

A

Left with 6 samples - pellets 1-4, final supernatant and the beginning mixture

Next, need to test all the samples for the activity of interest (eg. enzymatic activity if an interested in enzyme)

19
Q

What is the process of DENSITY-BASED cell centrifugation?

A

Use a sucrose gradient:
1) Spin the sample, form a gradient of sucrose concentration (high at bottom, low at top)

2) Sample sediments at different levels within the sucrose gradient
3) Empty the sample from the tube at the bottom and collect the liquid

20
Q

After running DENSITY-BASED centrifugation, what are you left with?

A

Fractions containing different samples of the liquid and the sediment

21
Q

In DENSITY-BASED centrifugation, how do the sample sediments settle in the sucrose?

A

At different concentrations of sucrose, depending upon where the sediments reach equilibrium with the sucrose

(Dense particles at the bottom, where the sucrose is concentrated. Light particles at the top, where the sucrose is at lower concentration)

22
Q

How is density-based centrifugation better than differential centrifugation?

A

Can separate out components in ONE round of centrifugation

23
Q

Describe the process of size-exclusion gel-filtration chromatography

A

1) Fill the column with porous beads (have holes in with a CONTROLLED SIZE)
2) Pour sample through

3) Small proteins enter bead - are slowed down
Larger proteins do not enter the bead (are EXCLUDED) - have a shorter path and travel faster

4) Collect fractions of the liquid which come out of the column over time (keeping the fractions separate)

24
Q

Why is buffer continuously added to the top of the column, during liquid chromatography?

A

To carry the sample through the column

25
Q

Describe the process of affinity chromatography

A

1) Covalently fix a substrate of the enzyme of interest to the bead - acts as the BAIT
2) Target enzyme binds to the bait, whilst the other proteins pass through the column
3) Check the fractions for protein until there is no protein present in the fractions
4) Add and eluting solution to the column, which breaks the specific interactions between the protein and its substrate
5) Check that the protein of interest has the desired activity and is still functioning (using electrophoresis, western blotting etc)

26
Q

What does affinity chromatography rely on?

A

Tight interactions

27
Q

In affinity chromatography, what is the ‘flow through’?

A

The proteins which do not bind to the bait on the beads (NOT the enzymes of interest)

28
Q

What can be added to the column in affinity chromatography to ‘elute’ the protein of interest from the beads?

A

A very high concentration of the substrate (which is attached to the beads)

The protein of interest will bind to this high concentration, rather than on the beads

29
Q

Describe the process of ion exchange chromatography

A

1) Use 2 different types of beads:
DEAE (have positive charge)
CM (have negative charge)

2) Pour solution through
3) Rinse with solvent

4) Slowly increase SALT CONCENTRATION - compete fo the ionic interactions between the proteins and the beads.
ELUTES the protein from the beads

5) Last proteins to come off will be proteins with a very high charge
6) Collect fractions and check these fractions for the desired activity (using electrophoresis, western blotting etc)

30
Q

What is the 3 step protein purification?

A

Multiple steps of purification using chromatography:
1) Ion-exchange

2) Gel filtration
3) Affinity

Results in protein purity increasing after each step

31
Q

What is the process of SDS-PAGE?

A

1) Gel box with 2 chambers:
Upper has negative charge
Lower has positive charge

2) Current through the gel, which connects the upper and lower chambers
3) Apply the sample in the wells at the top of the gel
4) Proteins travel to the negative charge, with smaller proteins travelling faster
5) Proteins represented as bands on a gel

32
Q

What is SDS-PAGE?

A

A type of gel electrophoresis, where Protein migration through the gel in the presence of SDS page is proportional to molecular mass

33
Q

In SDS-PAGE, what does the SDS do, along with heat and why is this needed?

A

Unfolds the proteins and gives them a negative charge, proportional to the size of the protein

Needed as protein folding, modifications and adhesion can affect migration through the gel

34
Q

In SDS-PAGE, what does beta-mercaptoethanol do?

A

Breaks the disulphide bridges between proteins - allowing them to migrate effectively through the gel

35
Q

What is dimensional gel electrophoresis and what does it form?

A

2 stages of electrophoresis:
1) ISOELECTRIC FOCUSSING - separates proteins by charge

2) SDS-PAGE - separates proteins on size

Produces a ‘fingerprint’ of the original protein sample

36
Q

What is dimensional gel electrophoresis used in?

A

Proteomics (analysis of all the proteins in a system)

37
Q

What is the process of isoelectric focussing, used in dimensional gel electrophoresis?

A

1) Gel has a STABLE pH GRADIENT
2) Add sample to the gel
3) Proteins move through the gel and stop when they reach equilibrium with that pH
4) Proteins form a line - dependant on how positively or negatively charged they are

38
Q

In isoelectric focussing, why do proteins stop moving through the gel?

A
  • They reach a pH which represents their charge
  • Equilibriate with this pH and become uncharged
  • Can no longer migrate through the gel (have no charge)

HIGH pH = -ve
LOW pH = +ve

39
Q

How can the proteins on the gel (separated by gel electrophoresis) be further analysed?

A

Using ANTIBODIES in WESTERN IMMUNBLOTTING

40
Q

What must occur before western immunoblotting can take place and why? (after gel electrophoresis)

A

Proteins must be transferred from the gel on to a protein binding membrane, using an electric current

As, in a gel - proteins are mobile and can move/diffuse away from their position

41
Q

Describe the process of western immunoblotting?

A

Uses 2 antibodies

1) Proteins electrophoretically transferred to a membrane, from the gel
2) Membrane Incubated with specific antibody
3) Use chemicals which are converted to colour or light

42
Q

Describe the process of mass spectrometry

A

1) Treat an isoloted protein with Trypsin (protease)
2) Separate the peptides (broken parts of the isolated protein)
3) Peptide ionisation
4) Ions are separated in the mass spectrometer according to their mass-to-charge ratio and detected in proportion to their abundance
5) Produces peaks on the graph with different peptides - each with unique mass

43
Q

Which 3 amino acids can be phosphorylated and why?

A

Serine
Threonine
Tyrosine

Because they have hydroxyl (-OH) groups