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Research Methods in Biochemistry > Protein Gel Electrophoresis > Flashcards

Protein Gel Electrophoresis Flashcards

1
Q

What are some of proteins crucial roles in biological processes ?

A
  • Transport and Storage
  • Mechanical support
  • DNA/RNA metabolism
  • Enzyme catalysis
  • Control of growth and differentiation
  • Immune response
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2
Q

What do proteins differ in ?

A
  • Size
  • Shape
  • Charge
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3
Q

What is Electrophoresis ?

A

This is the process by which large charged molecules travel through a matrix under the influence of a uniform electric field

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4
Q

What does the matrix act as ?

A

It acts as molecular sieve to aid separation

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5
Q

What are some examples of these matrix ?

A

Polyacrylamide gel, agarose gel, paper and cellulose

Polyacrylamide gel electrophoresis (PAGE)

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6
Q

How is polyacrylamide gel formed ?

A

By polymerisation of acrylamide

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7
Q

Explain PAGE ?

A
  • Mesh-like fiber network in three dimension

- Creates an environment that allows different sized proteins move through the gel at different rates

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8
Q

How does PAGE separate proteins ?

A

According to their molecular weight

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9
Q

However, proteins have different ?

A

They are different shapes, and have different charges

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10
Q

What does SDS-PAGE stand for ?

A

Sodium Dodecyl Sulphate Poly-Acrylamide Gel Electrophoresis

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11
Q

What is the aim in an experiment involving SDS-PAGE ?

A

To separate proteins according to their size i.e. chain length

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12
Q

What is the problem ?

A

Proteins have secondary, tertiary or quaternary structure → wide range of shapes and charges

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13
Q

So what is the solution ?

A

Use SDS to convert all proteins to the same shape and mask net charges

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14
Q

What does an Anionic detergent do ?

A
  • Disrupts non-covalent interactions (hydrogen bonds, hydrophobic interactions) thus unfolding the protein
  • Binds most proteins in a constant fashion (about 1.4 grams of SDS per gram of protein)
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15
Q

So, what does this ultimately do ?

A
  • Masks net charge of the protein by forming large anionic complexes
  • Minimises differences based on secondary or tertiary structure
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16
Q

Why are reducing agents added to do ?

A
  • Break disulphide bonds

- Detach subunits to ensure a separation based only on molecular weight

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17
Q

So what does this ultimately to do the proteins ?

A
  • Proteins have an homogenous negative charge
  • Move to cathode when electric field is applied
  • Larger proteins are more retarded.
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18
Q

Gel has two layers that differ in their acrylamide concentration and pH. Explain them ?

A
  • Stacking gel:
    low concentration acrylamide (4%), low pH (6.8)
  • Resolving gel:
    higher concentration of gel (10-20%) and high pH (8.8, same as tank)
19
Q

What needs to be done so all proteins all enter the separation gel at the same time ? resulting in ?

A

Concentrate proteins in a narrow band in the interface between gels
This will result in a greater resolution of bands

20
Q

One acrylamide concentration only separates a fixed range of protein sizes: hence ?

A

Use a gradient for a broader range of separation

21
Q

The proteins migrate through ?

A

An increasing percentage of acrylamide (e.g. 4-20%)

22
Q

Thus the gel becomes increasingly ?

A

Restrictive for the proteins to migrate

23
Q

Most commonly used in ?

A

Lab, commercial pre-cast gels

24
Q

Small proteins run too fast in ?

A

Low percentage gels

25
Q

Gradient gels can separate?

A

A broader range of protein sizes

26
Q

Once the gel is run we need to visualise the proteins:

A
  • Fix (immobilise) the proteins with a weak acid: proteins become insoluble and are thus prevented from diffusing during staining
  • Stain with Coomassie blue (or similar) dye to make protein bands visible
  • Then destain to remove excess dye
27
Q

Why use Non-denaturing gel electrophoresis (native-PAGE) ?

A

Separation according to:

  • Size
  • Net charge
  • Shape of native structure
28
Q

Native vs Denaturing gel run ?

A

In Native gel - All four levels analysed as secondary, tertiary and quaternary structure maintained
However, denaturing gel (e.g. reducing SDS-PAGE) - Primary structure analysis only happens

29
Q

What is Western blotting used for ?

A

Technique for identifying a particular protein using antibodies after electrophoretic separation in a gel and transfer to a membrane.

30
Q

Explain the three steps ?

A
  1. Separate proteins using SDS-PAGE
  2. Transfer into nitrocellulose membrane
  3. Stain and visualisation using antibody against protein of interest
31
Q

Why and how is two-dimensional gel electrophoresis used ?

A
  1. Thousands of proteins in cells: too many to separate by SDS-PAGE gel alone
  2. Use two dimensions, to separate the proteins on the basis of:
    - isoelectric point in the first dimension: isoelectric focusing
    - molecular weight in SDS-PAGE in the second dimension
32
Q

Low pH ?

A

Acidic groups neutralised

33
Q

High pH ?

A

Basic groups neutralised

34
Q

Change the pH, and what changes ?

A

The net charge changes

35
Q

What is the isoelectric point (pl) ?

A

pH at which the protein is neutral

36
Q

When is this different and what does this depend on?

A
  • Different for different proteins

- Depends on amino acid composition

37
Q

At all pH values other than their pI, proteins will be?

A

Charged

  • at low pH, the protein is positively charged
  • at high pH, the protein is negatively charged
38
Q

Proteins accumulate at a pH region that corresponds to ?

A

Their pI, where they have no net charge and migration ceases.

39
Q

Result: proteins focused into ?

A

Sharp stationary bands with each protein positioned at its pI.

40
Q

At the isoelectric point the protein has no ?

A

No net charge and therefore no longer migrates in the electric field; for the protein shown the isoelectric is 6.5

41
Q

Explain Protein identification ?

A
  • (Western blotting or)
  • Cut protein band out of gel
  • Digest with protease to make peptides
  • Identify by mass spectrometry
42
Q

What is Relative mobility (Rf ) ?

A

This is the distance migrated by a band divided by the distance migrated by the dye front (i.e. a small negatively-charged molecule).

43
Q

How can we measure Relative Mobility of Protein Bands ?

A
  • Related to the protein’s molecular mass
  • Run a set of protein standards of known molecular mass
  • Draw a standard curve of log (mass) versus relative mobility
  • Estimate apparent mass for unknown proteins

Research Methods in Biochemistry (3 decks)

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