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MSF1- Em > Chromatography Methods > Flashcards

Chromatography Methods Flashcards

1
Q

What is are the most commonly used adsorption/desorption techniques?

A

Ion exchange and affinity chromatography

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2
Q

What is ion exchange chromatography?

A

Separates proteins according to their charge and isoelectric point. Groups attached to beads in the column carry the charge. Polyanion (CM) is used for cationic exchange. Polycation (DEAE) is used for anionic exchange.

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3
Q

What are the limitations of ion exchange chromatography?

A

Proteins can have similar charges.

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4
Q

How is a buffer chosen in ion exchange?

A

Choose a buffer at a pH away from the pI of the protein. If the protein has a net negative charge, choose buffer above its pI. If the protein has a net positive charge, choose a buffer below its pI.

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5
Q

How is a column chosen in ion exchange?

A

It must be of opposite charge to the protein.

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5
Q

What is the nature of the solid phase in ion exchange?

A

Carbohydrate based. Hydrophilic to minimise non-specific interactions. Highly porous to avoid size exclusion. Rigid to optimise compression and made into fine particles for optimal flow.

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6
Q

Why might a weaker acid/base (CM/DEAE) be used for purification of highly charged proteins?

A

Highly charged proteins may bind too strongly to columns made of strong acids/bases (SP/QAE) and may be hard to elute.

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6
Q

Why does the buffer need to be chosen carefully when using a weak acid column?

A

Need to make sure the buffer doesn’t ionise the column- less able to specifically bind proteins.

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6
Q

How does ionic strength affect binding in ion exchange?

A

Small counter ions compete for charge on proteins and column, increases Kd. Protein has to displace counterions in order to bind to the column.

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6
Q

What are the typical conditions for ion exchange?

A

Start with buffer 1pH away from pI and ionic strength of 50-100mM. Salt concentration is then slowly increased.

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7
Q

Why might the quoted column capacity be inaccurate?

A

Column capacity depends on Mw of protein- quoted capacity may be incorrect for large proteins.

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7
Q

What happens if the ion exchange column is overloaded?

A

May get separation by size instead of charge.

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8
Q

Why might the pH of the column surface be different to the pH of the buffer in ion exchange?

A

H+ are excluded by positively charged beads and concentrated at negatively charged beads.

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9
Q

How can ion exchange be eluted using ionic strength and what is the advantage of doing this?

A

Increasing salt concentration, means column can be buffered at one pH.

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13
Q

What is chromatofocussing?

A

High resolution ion exchange method- separates proteins according to charge. Optimises the self-sharpening effect but is still limited by diffusional broadening.

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14
Q

What is the difference between chromatofocussing and ion exchange?

A

Ion exchange eluted using a changing salt concentration, chromatofocussing uses a pH gradient.

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14
Q

What type of buffer is needed for chromatofocussing?

A

A buffer with different ionisable groups at different pIs- use an ampholyte solution.

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15
Q

What is the order of elution in chromatofocussing?

A

Proteins tend to elute in order of their pIs, with proteins that are furthest from their pI eluting last.

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15
Q

How does chromatofocussing enhance the self sharpening effect?

A

One end of the column is at a pH above the pI of the protein and the other is at a pH below the pI of the protein (depends on proteins net charge). Forms a pH gradient either side of the pI throughout the column. Protein rebinds at the bottom of the column to allow the trailing edge to catch up.

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19
Q

When is hydrophobic interaction chromatography used and why?

A

Used if ion exchange doesn’t work because it is unlikely that proteins with the same charge will also have the same hydrophobic surface.

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20
Q

What is hydrophobic interaction chromatography?

A

Separates according to surface hydrophobicity, based on hydrophobic interactions between the target protein and the solid phase.

21
Q

What is the nature of the solid phase in HIC?

A

Carries bound aryl/alkyl groups and must be hydrophobic enough to the outcompete self interaction of the protein. Examples are phenyl (moderate hydrophobicity) and octyl (strong hydrophobicity).

22
Q

Describe the method used in HIC.

A

Protein loaded in high salt conditions. Protein eluted using a decreasing salt gradient.

23
Q

Why is the protein mixture loaded in high salt conditions in HIC?

A

Promotes the salting out effect and the protein binds to the column.

24
Q

How can highly hydrophobic proteins be eluted in HIC?

A

Using an organic solvent and water mixture, e.g. Ethylene glycol or methanol.

25
Q

What is reverse phase chromatography and when is it used?

A

Similar to HIC but uses a more hydrophobic ligand on the column. Higher resolution method. Used for peptides and carbohydrates.

26
Q

Why can reverse phase often not be used to separate proteins?

A

Proteins are denatured during the process and reverse phase can therefore only be used for proteins that can be refolded after separation.

27
Q

What conditions are used in reverse phase?

A

High pressure with organic solvent (acetonitrile or 0.05%-0.1% TFA)

28
Q

Give examples of ligands used on the column in reverse phase.

A

C18 or biphenyl

29
Q

What is affinity chromatography?

A

Based on the affinity of the protein of interest for another factor immobilised on the column.

30
Q

What factors need to be considered when designing an affinity chromatography experiment?

A

Non-specific binding at high protein concentrations. IEC/HIC effects of the chosen ligand. Lower binding capacity of a ligand when immobilised.

31
Q

Give a disadvantage of affinity chromatography.

A

Expensive, not suitable for large scale productions.

32
Q

Give the types of ligands that can be used in affinity chromatography columns.

A

Substrate analogues/inhibitors, cofactors, lectins, dyes, antibodies, fusion proteins with affinity tags.

33
Q

How is a His tag used in affinity chromatography?

A

Add His tag to the protein N terminus, protein binds to Nickel ions on beads, adding a low concentration of imidazole removes low affinity binding proteins, adding a high concentration of imidazole removes high affinity binding proteins.

34
Q

How can GST be used in affinity chromatography?

A

Added to protein N terminus with a cleavage site. Elute using gluthathione.

35
Q

Why must a cleavage site be added when using a GST fusion protein?

A

GST is a large tag and must be cleaved prior to NMR and XC analysis.

36
Q

What is an advantage of using GST?

A

Enhances solubility of the target protein.

37
Q

When is GST most commonly used?

A

During pull down assays.

38
Q

What is a pull down assay?

A

A type of affinity chromatography used to purify complexes. One molecule is indirectly bound to the column via another molecule which interacts with the column.

39
Q

How are ligands attached to the column in affinity chromatography?

A

The carbohydrate based matrix is activated by cyanogen bromide. Nucleophilic substitution reaction between the matrix and the spacer molecule. Reaction between the spacer and the ligand immobilises the ligand to the column.

40
Q

What is the advantage of immobilising the ligand to the column via a thiol linkage?

A

The reaction is reversible and the column in recyclable.

41
Q

How are protein eluted in affinity chromatography?

A

Via competition with another ligand in solution (used for lectins and fusion proteins) or using ionic strength (used in immunoaffinity).

42
Q

What is gel filtration chromatography?

A

Separates according to size. Liquid-liquid phase.The column is packed with porous beads. Small molecules can pass through pores in beads so move through the column more slowly. Large molecules pass through the spaces within the matrix so pass through the column more quickly and are eluted first.

43
Q

Why are recoveries from gel filtration chromatography high?

A

There are no interactions between the sample and the column.

44
Q

What is high performance liquid chromatography?

A

HPLC allows high resolution separation of proteins. Uses very high pressure to force the proteins through the column. Separates proteins by size.

45
Q

When is gel filtration usually used?

A

After adsorption/desorption to concentrate the target protein.

46
Q

What is a disadvantage of gel filtration chromatography?

A

It has limited capacity.

47
Q

Describe the nature of the solid phase in gel filtration.

A

Porous with a wide range of pore sizes. Solid beads account for 1-5% of the column. Long, thin columns are best.

48
Q

What volume of the column is experienced by small proteins?

A

Total column volume, as they can be included in the porous beads.

49
Q

What volume is experience by large proteins in gel filtration?

A

Void volume as they are excluded from the porous beads.

50
Q

What is Kav?

A

The fraction of internal volume that is accessible to the protein- Kav = (Ve - Vo)/(Vt - Vo)

MSF1- Em (7 decks)

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