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BIOM 3581A > Lab 4 - Affinity Chromatography > Flashcards

Lab 4 - Affinity Chromatography Flashcards

1
Q

What are the tasks of this lab?

A
  • Laboratory Introduction
  • Equilibrium of Ni-NTA Agarose matrix
  • Affinity Chromatography-Batch Method
  • Washing and Elution of His6-PTEN Protein
  • Quantitation of Purification Yield by Bradford Assay
  • Purity Analysis by SDS/PAGE
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2
Q

What is affinity chromatography employed to do in this lab?

A

Purify PTEN-His from bacterial lysates

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3
Q

What is the principle of affinity chromatography based on?

A

The exploitation of the specific, reversible binding between the protein of interest and a specific molecule immobilized on an inert support

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4
Q

What does the affinity chromatography technique have to offer?

A

High selectivity, hence high resolution, and high binding capacity for the protein of interest

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5
Q

Why is affinity chromatography a unique purification technology?

A

It is the only technique that enables the purification of a biomolecule based on its biological function or individual chemical structure

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6
Q

Purification that would otherwise be time-consuming, and complex using other techniques can often be easily achieved utilizing affinity chromatography in ____

A

a single step

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7
Q

What can affinity chromatography be used for?

A

To separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in high volumes of crude sample, and to remove specific contaminants

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8
Q

The biological interactions between ligand and target molecules can be a result of what?

A

Electrostatic or hydrophobic interactions, van der Waal’s forces and/or hydrogen bonding

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9
Q

How would the target molecule be eluted from the affinity matrix?

A

Interactions are reversed. Either specifically using a competitive ligand or non-specifically by changing the pH, ionic strength, or polarity

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10
Q

What does successful affinity purification require?

A

A biospecific ligand that can be covalently attached to a chromatography matrix

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11
Q

What must the coupled ligand retain?

A

Its specific binding affinity for the target molecules

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12
Q

After washing away unbound material, why must the binding between the ligand and target molecule be reversible?

A

To allow the target molecules to be removed in an active form

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13
Q

What can be used as a ligand to purify its respective binding partner?

A

Any component

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14
Q

What are some typical biological interactions frequently used in affinity chromatography?

A
  • Enzyme; Substrate analogue, Inhibitor, Cofactor
  • Antibody; Antigen, Virus, Cell (epitopes)
  • Lectin; Polysaccharide, Glycoprotein, Cell surface receptor
  • Nucleic acid; Complementary base sequence, Histones, Nucleic acid polymerase, Nucleic acid binding protein
  • Hormone or Vitamin; Receptor, Carrier protein
  • Metal ions; Poly (His) fusion proteins
  • Glutathione; Glutathione-S-transferase, GST fusion protein
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15
Q

What is the process of affinity chromatography?

A

1) Affinity matrix is equilibrated in binding buffer
2) Sample is applied under experimental conditions that favour specific binding of the target molecule
3) Target protein is recovered by changing the condition to favour elution of the bound molecule
4) Affinity matrix is re-equilibrated in binding buffer

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16
Q

What is the matrix?

A

An inert support to which a ligand can be directly or indirectly coupled

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17
Q

What are the properties required for an efficient and effective chromatography matrix?

A
  • Extremely low, non-specific absorption is essential since the success of affinity chromatography relies on specific interactions
  • Hydroxyl groups on the sugar residues are easily derivatized for covalent attachment of a ligand, providing an ideal platform for the development of affinity media
  • An open pore structure ensures high capacity binding even for large biomolecules since the interior of the matrix is available for ligand attachment
  • Good flow properties for rapid separation
  • Stability under a range of experimental conditions such as high and low pH, detergents, and dissociating agents
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18
Q

Where is the binding site of a target protein often located?

A

Deep within the molecule and an affinity medium

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19
Q

How is an affinity medium prepared?

A

By coupling small ligands, such as enzyme cofactors, directly to the matrix

20
Q

Why might low binding capacity be exhibited?

A

Steric interference (i.e., the ligand is unable to access the binding site of the target molecule)

21
Q

Under what circumstances are “spacer arms” used?

A

When low binding is inhibited

22
Q

How are spacer arms used to facilitate effective binding?

A

They’re interposed between the matrix and the ligand

23
Q

How must spacer arms be designed?

A

To maximize binding but to avoid non-specific binding effects

24
Q

When can the improvement of using a spacer arm be seen?

A

In purification

25
Q

What do all methods of eluting a protein involve?

A

The weakening of the interaction between the functional group on the matrix and your protein

26
Q

What are the basic methods for eluting a protein?

A

1) Change buffer composition
2) Change pH
3) Competition for binding with target
4) Add substance which competes for binding to the immobilized ligand (functional group)

27
Q

What is the most common method of affinity chromatography and what will be used in this lab?

A

4) Add substance which competes for binding to the immobilized ligand (functional group)

28
Q

How can the purification of recombinant proteins often be simplified?

A

By incorporating a peptide or protein tag of known size into a protein of interest

29
Q

Why are “tags” used?

A

Due to their high affinity for immobilized column support as well as providing a marker for expression and facilitating detection of the recombinant protein

30
Q

What are the two most commonly used tags?

A

Glutathione-S-transferase (GST) and 6 x histidine residues (His)6

31
Q

For this lab, what does the fusion protein contain and where is it located?

A

Contains 6 x histidine residues (His)6 and is located on the C-terminus of PTEN

32
Q

What are some of the characteristics of the (His)6 tag?

A
  • pH 8.0
  • Uncharged
  • Relatively small and therefore does not generally affect secretion, compartmentalization, or folding of the fusion protein within the cell
33
Q

How can proteins and peptides containing amino acids that have an affinity for metal ions be separated?

A

Using immobilized metal affinity chromatography (IMAC)

34
Q

How are the metals immobilized onto a chromatographic medium?

A

By chelation

35
Q

How is this IMAC matrix made?

A

By coupling a metal chelate forming ligand (iminodiacetic acid) to agarose

36
Q

Before use, the matrix is loaded with a solution of what?

A

Divalent metal ions such as Ni2+, Zn2+, Cu2+, Ca2+, Co2+, or Fe2+

37
Q

What is the binding reaction with the target protein dependent on?

A

pH

38
Q

How is the bound sample most commonly eluted?

A

By reducing the pH and increasing the ionic strength of the buffer or by including EDTA or imidazole in the buffer

39
Q

In this lab, what will be used for the isolation of His-PTEN and how will elution be performed?

A

Using a nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrix for the isolation of His-PTEN and elution will be performed using imidazole buffer

40
Q

What are the required lab results?

A

1) Bradford assay results and protein concentrations
2) Electronic image of your Coomassie Brilliant Blue stained gel

41
Q

How is the Ni-Agarose matrix equilibrated?

A

1) 15 ml tube containing 200 ul of Ni-NTA agarose slurry was centrifuged at 4100 rpm for 2 mins
2) Supernatant removed and transferred to waste
3) 6 mL ddH2O added to slurry and mixed by inversion to remove traces of ethanol
4) Matrix centrifuged at 4100 rpm for 2 mins
5) Supernatant removed and transferred to waste
6) 6 ml Wash Buffer added to tube containing matrix slurry and mixed by inversion then centrifuged at 4100 rpm for 2 mins
7) Supernatant removed and transferred to waste
8) Step 6 and 7 repeated

42
Q

What is the protocol for affinity chromatography-batch method?

A

1) Place 15 mL soluble lysate of His6-PTEN on ice
2) Add about 8 mL of His6-PTEN lysate to 15 mL tube of equilibrated Ni-NTA agarose matrix
3) Incubate tube with lysate and matrix for 1.5 hours at 4 degrees C

43
Q

What should be done while your sample is incubating?

A

Make one SDS-PAGE gel for purification analysis by following lab 3 protocol for gel preparation

44
Q

What is the protocol for washing His6-PTEN?

A

1) Centrifuge sample and matrix in swinging bucket rotator at 4100 rpm for 2 mins
2) Transfer 1 mL of the supernatant into fresh 1.5 mL microcentrifuge tube labelled “Flow through” and place on ice
3) Discard remainder of supernatant as waste
4) Add 6 mL of Wash Buffer to matrix and invert 5 times to wash resin
5) Centrifuge at 4100 rpm for 2 mins
6) Transfer 1 mL of the supernatant into fresh 1.5 mL microcentrifuge tube labelled “Wash 1” and place on ice
7) Discard remainder of supernatant as waste
8) Repeat steps 4-7 three times but from the last wash, save and transfer 1 mL of the supernatant into a fresh 1.5 mL microcentrifuge tube labelled “Wash 4” and place on ice

45
Q

What is the protocol for eluting His6-PTEN?

A

9) Incubate the matrix with 150 uL of Elution Buffer on ice for 20 minutes (occasionally flick/tap the bottom of the tube to facilitate elution about every 3 mins)
10) Spin down the beads at 4100 rpm for 2 mins
11) Transfer 100 uL of the supernatant into a fresh 1.5 mL microcentrifuge tube labelled as “Elution 1”
*Do NOT aspirate the resin
12) Repeat steps 9-11 one more time and label the tube as “Elution 2”

46
Q

What is the protocol for protein quantification by performing Bradford Assay?

A

Determine and record the protein concentration of the various samples to be loaded on the SDS-PAGE gel - samples include Flow through, Wash 1, Wash 4, Elution 1, and Elution 2 by following lab 2 protocol for Bradford Assay

47
Q

What is the protocol for analyzing protein purity by SDS-PAGE?

A

1) Analyze the purification procedure by mixing 20 uL of each sample with 4 uL of the 6 x SDS loading dye
2) Boil samples in heating block set to 95 degrees C for 5 mins
3) Store samples in green rack at room temp until ready to load
4) GA will slowly load 5 uL of protein molecular weight ladder mixture to first well
5) Slowly load 20 uL of your protein samples into the prepared SDS-PAFE gel
6) Run gel with 150V for approximately 45 mins
7) Follow lab 3 protocol for staining and destaining gel

BIOM 3581A (17 decks)

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