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Fundamentals Of Living Cells > Chromatography > Flashcards

Chromatography Flashcards

1
Q

Give the definition of chromatography

A

Lab technique used to separate mixtures of substances into their components

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2
Q

Why is chromatography important?

A

Isolates one protein from thousands to study its properties

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3
Q

What are the two phases in chromatography?

A 
Mobile phases (solvent)
Stationary phase (paper)
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4
Q

What are the types of properties of proteins that chromatography utilises?

A

Charge distribution
Molecular size
Solubility
Binding properties

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5
Q

What is salting in?

A

When low concs of salt are added the protein solubilities increase

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6
Q

How do salt molecules stabilise protein molecules?

A

Decreasing the electrostatic energy between the protein molecules, which increase the solubility of proteins

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7
Q

How do salt molecules increase the solubility of proteins?

A

The salt molecules compete with the protein molecules in binding with water

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8
Q

Why do protein molecules tend to associate with each other instead of salt molecules?

A

Protein-protein reactions become energetically more favourable than protein-solvent reactions

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9
Q

How does column chromatography work?

A

The compound mixture moves along with the mobile phase through stationary phase and it separates depending on the affinity/degree of adhesion of each component

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10
Q

What does ion exchange chromatography (IEX) do?

A

Separates larger amounts of materials than gel filtration

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11
Q

What is the IEX stationary phase?

A

A polymer (matrix/resin) attached with charged group

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12
Q

What do the acidic groups of resin interact with in IEX?

A

Positively charged proteins and are called cation exchangers

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13
Q

What do cation exchangers bind to in IEX?

A

Proteins with positive charges

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14
Q

Give an example of cation exchangers in IEX

A

CM cellulose canon exchangers

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15
Q

What happens in IEX if groups are basic in nature?

A

They interact with negatively charged molecules called anion exchangers

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16
Q

What is an example of basic groups in IEX?

A

Diethylaminoethyl (DEAE) cellulose

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17
Q

How do cation exchangers work?

A
  • Get polymer beads with a negative charge
  • add protein/ pass through the beads
  • proteins move through the column
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18
Q

In IEX cation exchanges, how do proteins with a more negative net charge move?

A

Move faster and elite earlier

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19
Q

Give the steps in cation exchangers

A

-proteins bind to an ion exchanger
-wash column with buffer to elute proteins based on how big an affinity thy have
-

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20
Q

What do cation exchangers bind to in IEX?

A

Proteins with positive charges

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21
Q

What do anion exchangers bind to in IEX?

A

Proteins with negative charges

22
Q

What does a high salt elution buffer do?

A

Increases mobility of remaining bands so they can be eluted

23
Q

What are other names for gel filtration chromatography?

A

Size exclusion chromatography or molecular sieve chromatography

24
Q

What is gel filtration chromatography used for?

A

Widely used to purify biological molecules in complex samples like blood

25
Q

What is separation based on in gel filtration chromatography?

A

Size and shape

26
Q

What does the column consist of in gel filtration chromatography?

A

Porous beads of dextran or agarose: Sephandex and sephanese are commonly used commercial preps

27
Q

What happens if the proteins can’t fit inside the beads in gel filtration chromatography?

A

They just pass over and are eluted first

28
Q

What is the elution volume?

A

The volume of a solvent required to elute a given solute from the column

29
Q

When is gel filtration chromatography usually used?

A

Near the end of the purification process

30
Q

What is affinity chromatography?

A

Relatively simple and effective molecular technique which isolated antibodies, antigens, hormones or other proteins by taking advantage of their binding affinity for their respective ligand

31
Q

What does affinity chromatography require?

A
  • Beads matrix
  • solution containing substance to be isolated
  • ligand that binds specifically to the target proteins
  • a wash to elute non-bound substances
  • final wash with elution buffer containing a competitive ligand to elute the target protein
32
Q

What is affinity material made up of?

A

Inert support + spacer arms + ligand

33
Q

What must the first wash in affinity chromatography be?

A

Sufficient solute concentration, temp and pH to elute all unbound impurities

34
Q

What should the second wash in affinity chromatography contain?

A

Competitive ligand

35
Q

What is the second wash in affinity chromatography used for?

A

Remove competitive ligand by dialysis after its been eluted

36
Q

What does high performance liquid chromatography allow for?

A

The used of smaller profile solution for the column material, thus increasing the surface area

37
Q

What does a high surface area allow in high performance liquid chromatography?

A

Much better separation of samples based on hydrophobicity

38
Q

What is the role of the pump in high performance liquid chromatography?

A

Force the mobile phase through the column at a specific rule expressed in millilitres per minute

39
Q

What does the injector do in high performance liquid chromatography?

A

Introduces the sample into the flow stream of the mobile phase

40
Q

What is a typical sample volume of a high performance liquid chromatography?

A

5-20 micro litres

41
Q

In a chromatogram what does the area underneath the peak signals represent?

A

How much of the thing there is

42
Q

What are the advantages of high performance liquid chromatography?

A 
Speed
High resolution
Sensitivity 
Reproducibility 
Accuracy
Automation
43
Q

What are the disadvantages of high performance liquid chromatography?

A

Cost
Complexity
Coelution

44
Q

What is coelution

A

Sometimes there are two molecules each time with the same affinity for the mobile phase, so they elute at the same time?

45
Q

What are the two steps in 2D electrophoresis?

A

Isoelectric focussing

SDS-PAGE

46
Q

What does SDS-PAGE stand for?

A

Sodium dodecyl sulphate- polyacrylamide gel electrophoresis

47
Q

How does SDS-PAGE work?

A

Separates proteins by molecular weight so its based on the size of the proteins

48
Q

What does a western blot use?

A

Electrophoresis for protein seperation

49
Q

Where is the sample placed in elecrophoresis?

A

Onto the negative end of the polyacrylomide gel

50
Q

What happens after blotting in electrophoresis?

A

The membrane is blocked in order to prevent and unwanted membrane-blotter interaction to visualise the protein of interest

Fundamentals Of Living Cells (41 decks)

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