Gel Electrophoresis Flashcards

1
Q

What is gel electrophoresis ?

A

Defined as the separation of molecules based on migration in an electromagnetic field

Uses 2 electrodes - anode (+ve) and cathode (-ve) and
an electrolyte gel which serves as a conducting medium

The rate and direction of the particles moving within the gel will be dependent on their size and molecular charge

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2
Q

What can be separated ?

A

Anything which possesses ionisable groups which at a given pH in solution exist as an electrically charged species

E.g amino acids, nucleotides and nanoparticles

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3
Q

Describe the Agarose Gel Electrophoresis technique:

A

Molecules to be separated are pulled by an electrical field (negative to positive) through a gel containing
small pores - agarose

Molecules travel through agarose at a speed inversely related to their length

Smaller DNA molecule (less -ve charge) will travel further

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4
Q

Describe the gel age of agarose:

A

0.7% - 2.0% agarose

0.7% - good separation of large DNA fragments

2.0% - good separation of small DNA fragments

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5
Q

Describe the visualisation of agarose including advantages and disadvantages of new products:

A

Visualisation dyes can be added before, during or after the gel is run

Previously used Ethidium Bromide and UV light but now much safer molecules are used (less teratogenic and mutagenic)

Now use SYBER safe or GelRed®

Advantages –
relatively easy, cheap and fast to perform, can give you a rough size of product

Disadvantages – semi quantitative as you can’t determine the amount of product generated easily

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6
Q

Describe the Polyacrylamide Gel Electrophoresis (PAGE) technique :

A

Used to separate proteins or fragments of DNA <1 kb

Formed by the polymerisation of acrylamide + N,N -methylene-bis-acrylamide (the cross-linking agent)

Polymerization initiated by addition of ammonium persulfate + DMAP or TEMED

2 types;
- native non denaturing PAGE
- denaturing PAGE

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7
Q

What is native non-denaturing PAGE ?

A

Used to separate proteins/ nucleic acids based on their size, shape, and charge, without disrupting their natural structure or function

Mobility depends on protein charge & hydrodynamic size

Possible to recover proteins in their native state

Protein-protein interactions can be observed

Enzymatic activity of a protein can be assayed after separation

Resolution generally not as high as that achieved with SDS PAGE

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8
Q

What is denaturing PAGE ?

A

Disrupts 2°, 3°, and 4°structures – unfolds the protein

Use of sodium dodeycl sulphate (SDS) confers -ve charge to the proteins

Mobility depends on the size of protein - good for molecular weight determination

Higher resolution than non-denaturing PAGE

Determines relative abundance of major proteins in a sample

Protein-protein interactions or aggregations that occur in vivo won’t be observed

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9
Q

How can you improve resolution ?

A

Discontinuous systems - separating gel or stacking gel

Proteins are compressed and stacked in order of mobility
Stacking gel contains chloride ions while the electrophoresis buffer contains glycine ions
Leading ions - migrate through gel faster than protein sample
Trailing ions - slower than protein sample
Protein molecules are trapped in a sharp band between these ions

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10
Q

What is Isoelectric Focusing (IEF) ?

A

Technique for separating molecules based on their different isoelectric points

At pH below the pI, protein will have + charge
At pH above the pl, protein will have a -ve charge

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11
Q

What are IEF gels ?

A

Strips or columns of
polyacrylamide containing a pH gradient (IPG strip)
Proteins migrate according to charge until they reach portion of the strip where the pH corresponds to their pI
Net charge is 0 and migration stops
Native or denaturing conditions are both possible

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12
Q

Describe the combination of IEF with PAGE:

A

Combination of IEF with PAGE - 2D Electrophoresis
In the first dimension, proteins are separated based on their isoelectric point (pI), which is the pH at which a protein has no net charge by IEF

In the second dimension proteins are separated based on molecular weight by SDS-PAGE

ID by mass spectrometry
Used to separate complex mixtures

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13
Q

What are the advantages and disadvantages of 2D Gels;

A

Advantages;
- can resolve proteins that have undergone some form of post translational modification
- different gradient gels available for better resolution
- relatively inexpensive

Disadvantages;
- automated spot picking robots for MS/MS identification are very expensive
- can’t resolve very large proteins, very hydrophilic or very hydrophobic proteins

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14
Q

What is capillary electrophoresis ?

A

Used for separation of a variety of analytes of different sizes and charges - organic, inorganic ions, metal ions, drugs

Electrophoresis carried out in capillaries

Usually fused silica coated with polyimide

Use of a support medium (gel) is not necessary

Analysis carried out in free aqueous solution

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15
Q

Describe how capillary electrophoresis works:

A

Samples migrate along the tube due to;

Electrophoreticmigration (EM) - charged molecules move to oppositely charged electrode

Electroendosmosis (E) - Phenomenon due to presence of charged groups on surface of the support, a much stronger force than EM

All molecules move from the anode (+) towards the cathode (-) and are separated on basis of size and charge simultaneously

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16
Q

What are the advantages of capillary electrophoresis ?

A

High separation efficiency

High voltages to move the analytes;
faster = short analysis times

Good for forensic DNA analysis due to accuracy and speed

Low sample volumes - typically nanolitres (nl) –microliters (μl)

Hugely diverse range of applications

17
Q

What are the disadvantages of capillary electrophoresis ?

A

Sample sticking to capillary walls

Lack of separation or focus in peak, makes differentiation hard

Decreased species stability leading to new species peaks

Inconsistent retention time