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analytical test 2 > Gel Electrophoresis of Proteins > Flashcards

Gel Electrophoresis of Proteins Flashcards

1
Q

In denaturing protein Gel electrophoresis what is lost?

A

quaternary structure and biological activity

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2
Q

Denaturing Protein Gel electrophoresis is primarily done with what?

A

Sodium dodecyl sulfate (SDS)

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3
Q

What is SDS?

A

Sodium dodecyl sulfate (SDS) is a detergent

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4
Q

SDS has what structure?

A

Hydrocarbon tail (hydrophobic tail) and hydrophilic head

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5
Q

What does SDS do to folded proteins?

A

Unfolds protein

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6
Q

How does SDS interact with folded proteins?

A

The detergent interacts with folded protein by coating hydrophobic regions of the polypeptide conferring negative charges on them. This disrupts both subunit-subunit and protein-membrane interactions.

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7
Q

SDS disrupts what type of interactions?

A

subunit-subunit and protein-membrane interactions

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8
Q

The hydrophobic portion of SDS intercalates where in proteins?

A

the hydrophobic parts of the protein.

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9
Q

SDS “coats” proteins with a uniform layer of what?

A

Negative charges.

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10
Q

What does SDS PAGE stand for?

A

Sodium dodecyl sulfate- polyacrylamide gel electrophoresis.

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11
Q

SDS separates molecules how?

A

Based on M.W.

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12
Q

How can M.W be estimated my SDS-PAGE?

A

1) Calibrate curve of log molecular weight on the x-axis and Distance migrated on the Y-axis for molecules loaded into the SDS page of known molecular weight
2) Use linear relationship of graph and the equation it creates to find the M.W of the unknown sample molecules based on migration distances.

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13
Q

Why can proteins sometimes bind above-average or below average affecting migration distances?

A

amounts of SDS affects migration rate

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14
Q

Will Fibrous abd globular proteins show some differences in SDS PAGE?

A

It may but some structure may still be present

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15
Q

What type of proteins will appear larger than other proteins?

A

Conjugated proteins such as Phosphorylated proteins

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16
Q

Phosphorylated proteins are what type of proteins?

A

Conjugated

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17
Q

Electrophoresis using Urea is what type of electrophoresis?

A

Denaturing electrophoresis

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18
Q

Urea disrupts what structures?

A

secondary, tertiary and quarternary structure

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19
Q

What is Urea’s net charge?

A

Does not have one

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20
Q

How does Urea migrate in an electric field?

A

It doesn’t because it has no net charge.

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21
Q

In Denaturing electrophoresis of proteins using Urea proteins move how?

A

proteins migrate based on their intrinsic charge.

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22
Q

Urea disrupts what in DNA/RNA?

A

Hydrogen bonds between base pair

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23
Q

Denaturing electrophoresis of proteins using urea is extensively used for what?

A

DNA sequencing gels

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24
Q

Are disulfide bonds/bridge intermolecular or intramolecualr?

A

Can be either

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25
Q

Disulfide bridges are formed between what?

A

cysteine side-chain sulphydryl groups

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26
Q

What is an intrachain disulfide bond

A

Disulfide bond that occurs within a single polypeptide

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27
Q

What is an interchain disulfide bond?

A

Disulfide bond what occurs between different polypeptides.

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28
Q

Why are disulfide structures important?

A

Frequently maintain the protein in a particular shape and usually add to its stability.

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29
Q

How can dislufide bonds be reduced to sulphydryls?

A

by treatment with a reducing agent such as Beta-mercaptoethanol or dithiothreitol

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30
Q

What does Beta-mercaptoethanol do to disulfide bonds?

A

reduces disulfide bonds to sulphydryls.

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31
Q

What does dithiothreitol do to disulfide bonds?

A

reduces disulfide bonds to sulphydryls.

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32
Q

If a protein is a trimer that treating it with a reducing agent such as Beta mercaptoethanol it will do what?

A

Will disrupt the disulfide bonds and break the protein into its three subunits.

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33
Q

A protein is a trimer with two 25kDa and a 50kDa subunit if it is ran in the presence of a reducing agent such as Beta-mercaptoethanol than how many bands will be formed?

A

2; one at 25 kDa because all the subunits are separated by travel based on M.W and two subunits have the same M.W. The other band will be at 50kDa since this is the M.W of the third subunit

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34
Q

A protein is a trimer with two 25kDa and 50kDa subunits if it is ran Without the presence of a reducing agent such as Beta-mercaptoethanol than how many bands form?

A

One; At 100 kDa because 50+25+25 = 100, the protein moves based on M/W/ but the subunits are not broken apart but remain as a single protein.

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35
Q

After a polypeptide linked by disulfide bond is separated by reduction with dithiothreitol the polypeptide is followed with what? why?

A

alkylation to prevent reformation.

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36
Q

What is a common molecule used for alkylation?

A

Iodoacetate.

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37
Q

What does Diagonal electrophoresis do?

A

locates disulfide bonds within a single polypeptide subunit

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38
Q

Diagonal electrophoresis is typically done with what?

A

1) SDS-PAGE (-) and (+) DTT
OR
2) paper (-) and (+) performic acid

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39
Q

What is the first step in Diagonal Electrophoresis?

A

The protein is specifically cleaved into peptides under conditions in which the disulfide bonds remain intact.

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40
Q

What is the second step of Diagonal electrophoresis after the protein is specifically cleaved into peptides under conditions in which the disulfide bonds remain intact?

A

The mixtures of peptides is applied to a corner of a sheet of paper and subjected to electrophoresis in a single lane along one side.

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41
Q

What is the third step in diagonal electrophoresis after the sample mixtrure of peptides is electrophoresed in a single lane along one side?

A

The resulting sheet is exposed to vapors of performic acid which cleaves disulfides and converts them into cysteic acid residues.

42
Q

What happens in diagonal electrophoresis using SDS-PAGE when proteins are exposed to vapors of performic acid?

A

Cleaves disulfide bonds and converts them into cysteic residues. Peptides originally linked by disulfides are now independent and more acidic because of the formation of an SO3- group.

43
Q

What is the fourth step after disulfide bonds are cleaved from perormic acid in diagonal electrophoresis?

A

The mixture is subjected to electrophoresis in the perpendicular direction under the same conditions as those in the first electrophoresis.

44
Q

How does diagonal electrophoresis help locate disulfide bonds?

A

Peptides that are devoid of disulfides will have the same mobility after the addition of performic acid as before and will be located on a single diagonal line. While peptides whom did have disulfide bonds the newly formed peptides containing cysteic acid will usually migrate differently from their parent disulfide-linked peptides and therefore lie off the diagonal. These peptides can then be isolated and sequenced and the location of the disulfide bond can be established.

45
Q

How does a peptide devoid of disulfide bonds act in diagonal electrophoresis?

A

Peptides devoid of disulfide bonds will have the same mobility after performic acid as it did before thus be located on a diagonal line.

46
Q

How does a peptide with disulfide bonds act in diagonal electrophoresis?

A

Peptides with disulfide bonds treated with performic acid will now contain cysteic acid what will usually migrate differently from their parent disulfide-linked peptides and hence will lie off the diagonal.

47
Q

Does performic acid reduce disulfide bonds?

A

No it oxidizes the disulfide bonds.

48
Q

Chemical cross-linking analyses what?

A

quaternary structure

49
Q

Subunits of proteins are often held together by what?

A

weak, non-covalent interactions, especially hydrophobic interactions.

50
Q

What makes it difficult to study quaternary structure?

A

when subunit association is very unstable.

51
Q

What amino acid is abundant in proteins?

A

Lysines (~7%)

52
Q

Lysines have a chemically reactive what group?

A

E-NH2 group or E-NH3+

53
Q

Lysine e-NH2 groups of adjacent polypeptides may be crosslinked by what?

A

a bifunctional reagent such as dimethyl suberimidate.

54
Q

Crosslinked proteins will electrophase in SDS PAGE to an apparent mass wqual to what?

A

that of the sum of the cross-linked polypeptides.

55
Q

Crosslinking chemicals such as Dimethyl suberimidate are called what?

A

bifunctional reagents

56
Q

what polypeptides are crosslinked?

A

Polypeptides adjacent to each other in the quaternary structure will be covalently crosslinked if lysine and lysine distances fall within a maximum range.

57
Q

Isoelectric focusing is electrophoresis of proteins in what?

A

pH gradients

58
Q

In a pH gradient a protein will migrate unitl what?

A

it reaches pH at which its’ net charge is zero (pI of protein)

59
Q

The pH gradient is formed by what?

A

electrophoresing a mixture of small polyampholytes (or “ampholytes”)

60
Q

what are polyampholytes or “ampholytes”

A

small, multicharge of polymers with different PI’s.

61
Q

Isoelectric focusing can resolve proteins with differences in PI of what?

A

0.01

62
Q

Proteins differing by ____ net charge can be separated?

A

1

63
Q

Ampholytes are short polymers containing what groups?

A

Both NH2 groups and -COOH groups.

64
Q

When ampholytes are mixed together in the absence of electric field what occurs?

A

a single net pH results

65
Q

When ampholytes are mixed together in the presence of an electric field what occurs?

A

Individual ampholytes migrate to their PI values.

66
Q

In isoelectric focusing ampholytes act as what?

A

acts as buffers resulting in a pH gradient.

67
Q

During electrophoresis of proteins during isoelectric focusing amphyolytes move how at their PI?

A

Do not move since they have no net charge at their PI

68
Q

Ampholytes means a chemical with both a ____ and ____ group so _____ are ampholytes.

A

they have both positive and negative charged groups so technically proteins are also ampholytes.

69
Q

In isoelectric focusing (IEF) when placed in a pH gradient proteins with a positive net charge will migrate _____ while proteins with a negative net charge will migrate how?

A

In isoelectric focusing when placed in a pH gradient proteins with a positive net charge will migrate towards the cathode while those with a net negative charge will migrate towards the anode.

70
Q

Explain IEF experiment in a glass tube

A

The pH gradient is formed in glass tubes which are placed in an electrophoretic device. After IEF the gel is cut into thin slices and the pH of each slice is determined. From this a graph of mobility versus pH may be generated. From the mobility of protein band, pI of protein may be determined.

71
Q

Explain IEF experiment in flat-bed

A

The flat-bed format allows IEF standard proteins of known pI alongside sample protein. Comparison with standards allows determination of pI.

72
Q

From experiments of IEF a graph of what can be done allowing for what?

A

From this a graph of mobility versus pH may be generated. From the mobility of protein band, pI of protein may be determined.

73
Q

What is 2-Dimensional gel electrophoresis?

A

Isoelectric focusing + SDS-PAGE

74
Q

Why would 2-Dimensional gel electrophoresis be used?

A

It is unlikely that 2 polypeptides will have the exact same PI and molecular mass.

75
Q

The majority of polypeptides in a complex mixture will appear as what?

A

Distinct separate spots on a 2-D gel.

76
Q

What are the limitations of 2-Dimensional gel electrophoresis?

A

some proteins are expressed at very low levels and are not detectable when an extract is run on a 2-D gel.

77
Q

What helps with the limitation of 2-Dimensional gel?

A

silver-staining

78
Q

How many proteins can be resolved by 2-D gels?

A

more than 1,000 proteins

79
Q

A two dimensional gel electrophoresis coupled with mass spectrometric techniques and/or partial amino acid sequencing it is possible to do what?

A

Proteins can now be identified which use to be a former draw back.

80
Q

What are uses of two dimensional gel?

A

1) Compare 2-D spots in normal cells and cancer cells as well as some tissues
2) Compare 2-D spots in normal cells and gene-inactivated mutant cells
3) 2-D spots in cells in G1 phase vs. S vs. G2 vs M
4) 2-D spots in cells before and after exposure to X-rays

81
Q

Non-denaturing electrophoresis of Nucleic acids

A

DNA/RNA almost always run on agarose gels, horizontally

82
Q

Both proteins and DNA give approximately linear graphs of what?

A

log M.W versus mobility.

83
Q

Agarose gels pores are larger than in polyacrylamide gels so what?

A

so resolving similar-sized small DNA molecules is difficult.

84
Q

If a bp is less than 100 what gel should be used?

A

Polyacrylamide

85
Q

100 bp to 50,000 bp what gel should be used?

A

Standard Agarose gel

86
Q

More than or equal to 50,000 what gel should be used?

A

Pulsed Field Electrophoresis

87
Q

A chromosome seperation would be performed on what type of gel?

A

Pulsed Field Electrophoresis

88
Q

Unlike globular proteins nucleic acids have what?

A

large axial ratios

89
Q

What is an axial ratio?

A

ratio of longitudinal to transverse axes

90
Q

The nucleic acid is free to rotate through all possible orientations and therefore migrates as if it what?

A

has a very large molecular mass many times more than their actual mass

91
Q

Charge distribution ( negative phosphate)are similar for all nucleic acids whus separation is by what?

A

mass

92
Q

Rod like molecules like nucleic acids as well as ____ proteins have large axial ratio

A

fibrous

93
Q

What are the two models of how nucleic acids migrate through gels?

A

1) Biased-reptation model

2) Caterpillar model

94
Q

What is “reptation”?

A

like the side-to-side slithering motion of a snake.

95
Q

In Biased-reptation model DNA orientates how?

A

In the direction of the electric field and DNA enters the pore regardless of its molecular mass

96
Q

In caterpillar model DNA forms a compact mass which becomes retarded on an agarose fibre. It does what?

A

It extends into a U-shape around the fibre. The head explores new routes through the gel. The tail is pulled along behind the head until compact mass is again formed. The tail can act as head in the next cycle of extension and contraction. Migration of head region through the gel is independent of mass.

97
Q

Pulsed Field Gel Electrophoresis (PFGE) is used for what?

A

Used for very large DNAs.

98
Q

Pulsed Field Gel Electrophoresis (PFGE) requires what?

A

Special apparatus

99
Q

How does PFGE act?

A

DNA fragments are exposed to electric force which alternates in different directions as a result of electrical pulses. The DNA moves through the gel in response to this field. Small molecules move more quickly than large ones due to their shorter viscoelastic relaxation times.

100
Q

The principle of pulsed field electrophoresis large molecules are electrophoresing but what happens?

A

Becomes entangled in the gel and the field is briefly reversed allowing untangling. The normal direction of electrophoresis is resumed and the cycle is repeated over and over.

101
Q

Large DNA fragments more than 50,000 bp Shear easily therefore DNA must be purified carefully without what?

A

without vortexing or pipetting.

102
Q

Typically cells are lysed in an agarose gel plug which is then what?

A

placed in the well of the gel.

analytical test 2 (1 decks)

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