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Genomic and Proteomics > Chapter 6 isoelectric focusing > Flashcards

Chapter 6 isoelectric focusing Flashcards

1
Q

Objectives

A

Understand the principles of isoelectric focusing (IEF)
Understand how a pH gradient is formed
Understand advantages of immobilised pH gradients (Immobiline)
Understand the techniques used in IEF experiments and applications of IEF

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2
Q

Principles of IEF

A

An electrophoretic process, in which proteins are separated according to their isoelectric points

Regardless of the point of loading, proteins are “focused” to seek their isoelectric points
Isoelectric point (pI) is the pH at which a protein has a net charge of zero.
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3
Q

If the pH is less than pi

A

the protein becomes positively charged, which makes it attracted to the negative end of the strip, low pH

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4
Q

If the pH is more than pi

A

the protein becomes negatively charged, which makes it attracted to the positive end of the strip, high pH

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5
Q

How being amphoteric affects the protein

A

Depending on the pH of the environment, each protein can be overall positively or negatively charged or have zero net charge.

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6
Q

Relationship between charge and pH

A

Overall charge of a protein is dependent on the pH of the surrounding environment

pH > pi, proteins are negatively charged
pH < pi, proteins are positively charged
pH = pI, proteins have no charge

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7
Q

Why form pH gradient

A

a pH gradient needs to be established in order for proteins to seek out their isoelectric points

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8
Q

What are the 2 types of reagents used to generate a pH gradient?

A
  1. carrier ampholytes

2. immobiline reagents

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9
Q

Carrier ampholytes use

A

Used in conventional IEF

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10
Q

Immoboline reagents use

A

Used in immobilined pH gradient IEF

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11
Q

Carrier ampholytes principle

A

They are low molecular weight (400-1000Da) zwitterions at a certain pH and buffer at that pH

Synthetically-made molecules to comprise a range of pI values.

When an electric field is applied, carrier ampholytes will arrange themselves to build up a pH gradient.

Comes in a range of pI ranges to achieve optimal resolution.

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12
Q

Role of carrier ampholytes in IEF

A

form a pH gradient when added

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13
Q

Ampholytes and gel matrix

4 steps

A

Ampholytes are not well integrated with the gel matrix

  1. an ampholyte solution is incorporated into a gel
  2. a stable pH gradient is established in the gel after application of an electric field
  3. protein solution is added and electric field is reapplied
  4. after staining, proteins are shown to be distributed along pH gradient according to their pi values
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14
Q

2 disadvantages of conventional IEF system

A
  1. carrier ampholytes are more mobile than proteins
    , thus IEF needs to be continued after pH gradient is set up
  2. need for optimization of focusing time after pH gradient is set up, otherwise the pH gradient collapse due to cathodic drift
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15
Q

cathodic drift problem

A

cathodic drift, where the pH gradient decrease over time, may occur if a gel is focused too long.

Cathodic drift is observed as focused protein migrating off the cathode end of the gel.

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16
Q

Electro-osmotic flow

A

Movement of water (H3O+) to cathodic end, carries with it basic ampholytes and proteins

A result of prolonged IEF
More than standard duration of usually 3hours

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17
Q

Main cause of cathodic drift

A

Electro-osmotic flow

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18
Q

How to prevent pH gradient collapse

A

What if the pH gradient can be pre-formed and immobilized on a gel?

19
Q

How to immobilize pH gradient

A

Immobiline (IPG)

20
Q

Immobiline (IPG), use of acrylamide monomer

A

Use of weak acidic and basic buffering reagents, covalently bound to an acrylamide backbone

Mixing different proportions of the weakly acidic and basic buffering reagents result in “carrier ampholytes” buffering at different pH.

Titration of the weakly acidic and basic buffering reagents set up a pH gradient during gel casting.

The backbone of the reagents is an acrylamide monomer, allowing polymerisation to take place with the gel.

Polymerization immobilizes pH gradient

21
Q

Immobiline dry strip

A

commercially available
can choose pH range
can choose length of the strip

22
Q

immobline reagent

A

acidic and basic buffering reagent that we use to immobilize the pH gradient

23
Q

what happens when immobiline reagents are integrated with IEF gel

A

Incorporation of different amounts of amine and carboxylic groups covalently bound in result in different localised pH environments, thus create pH gradient

24
Q

2D-PAGE using IPG DryStrip

A

the stacking gel region is not the usual stacking region, it is tier spaced, allow us to transfer the strip once we completed the first dimension

carry out the normal SDS-PAGE, proteins will now be resolved based on the molecular weight

25
Q

The stacking gel in 2D-PAGE using IPG DryStrip

A

Stacking gel (%T=4-6)

IPG DryStrip (T=4%,C=3%) replaces the stacking gel, (enable loading of proteins at high concentrations)

26
Q

The resolving gel in 2D-PAGE using IPG Dry Strip

A

Resolving gel (%T fixed between 10-15)

Separates proteins by MW

27
Q

There are different types of dry strips

A

vary in pH ranges and length, depend on sample we work with

28
Q

Choice of pH range

A

depends on sample, analysing protein from cell lystae, using broad pH, better separation of all proteins

separating proteins characterized by pH and Pi, narrow will more suitable

non-linear drystrip basically allow more even distribution of proteins of interest

29
Q

broad pH range uses

A

Broad pH range gel strips allow separation of most protein mixtures from prokaryotic and eukaryotic sources.

30
Q

narrow pH range uses

A

Narrow pH range gel strips allow better resolution of proteins with a known pI range, especially those that do not resolve well on a broad range gel.

31
Q

Non-linear pH range uses

A

Gel strips with nonlinear (NL) pH ranges allow a more even distribution of proteins along a specific pH range to maximise resolution.

32
Q

Round 1

A

Broad pH range for maximal resolution of all proteins

33
Q

Round 2

A

Narrow pH range to better resolve particular group of proteins

34
Q

2D PAGE

A

the blue streaks and spots correspond to stained proteins

use a narrow pH range subsequently after broad pH range to separate the proteins better

35
Q

Linear gel

A

Linear pH gradient over the whole pH range

36
Q

Non-linear gel

A

Increased resolution between pH5 to 7 with a non-linear pH gradient

It can separate proteins better. Proteins are much more well separated than when we use non-linear gel

37
Q

Estimating pi of individual protein spots

A

we can also use gel strip to determine pi of the respective proteins spots

measure length of strip

determine the distance of protein spot of interest

express the 2 above as a percentage

The pI of the protein spot can be estimated by relating the position of the protein in the 2D-PAGE gel to its original position in the Immobilline drystrip.

38
Q

Plot a graph displaying pH gradient of the strip vs the percentage of the gel

A

Read of the graph
y axis is pi range
% gel length is x axis
read off the 2

39
Q

immobiline in dry spots

A

Besides non-linear gel strip to improve the resolution of protein spots, use gel strip with overlapping ranges

40
Q

Improving resolution using “composite maps”

A

Protein separation is not too ideal when it moves to the right, a bit cluttered. If we are interested in those proteins, run a gel from 4.5-5.5, allow region from 4.5 to be better separated as compared to the original gel from 4 to 5

advantage of using overlapping pH ranges, help to improve the resolution

41
Q

2 steps principle of Improving resolution using “composite maps”

A

Creation of ‘composite maps’ with overlapping pH ranges.

Enables better resolution of low abundance proteins.

42
Q

How to determine length of drystrip to use

A

as we improve the length of the strip, the time to carry out the 1st dimension also increases

43
Q

Advantages as the length of strip increases

A

Sample loading capacity increases
Resolution of proteins increases
Number of spots detected increases

44
Q

Disadvantage as the length of strip increases

A

Focusing time increases, more time needed for focusing

Cost effectiveness decreases

Genomic and Proteomics (9 decks)

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