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BIOC 3622 - Biochemistry Lab > Spectrophotometric Determination of Protein Concentrations > Flashcards

Spectrophotometric Determination of Protein Concentrations Flashcards

1
Q

______ describes the technology that focuses on the interaction of biological materials with _____ and other forms of radiant energy whose quantum unit is the _____.

A

Biophotonics; light; photon

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2
Q

_____ is energy that comes from a source and can travel through material or space.

A

Radiation

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3
Q

Mnemonic to remember the electromagnetic spectrum?

What does it mean?

A

Raging Martians Invade Venus Using X-Ray Guns

A few ways to remember the order of the Electromagnetic radiation spectrum’s wavelength from low to high frequency, from lowest to highest in energy:

Radio waves, Microwaves, Infrared, visible light (Red, Orange, Yellow, Green, Blue, Indigo, Violet), Ultraviolet, X-rays, and Gamma rays.

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4
Q

Tissues and cells are composed of what 4 different biomolecules?

A

DNA, proteins, lipids, carbohydrates

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5
Q

In what 4 ways can light interact with biomolecules?

A

reflection, absorption, transmission, light scattering

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6
Q

The ________ is based on the absorption of light as a function of wavelength.

A

Bradford assay

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7
Q

As light passes through a material, _________ is absorbed, and each material absorbs _____ at a specific ______.

A

light energy; light; wavelength

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8
Q

The removal of these wavelengths from visible light gives the material its ______.

A

color

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9
Q

The removal of yellow wavelengths of light by the protein-dye complex at 595 nm makes the protein-dye complex _______, while the dye alone (without protein) absorbs light at 470 nm making the dye a ________ color.

A

blue; reddish-brownish

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10
Q

Nearly all biophotonic applications involve a light source that is passed through a target material and a ___________ that reads the ________ from the material.

A

detection sensor; light emission

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11
Q

A __________ has a light source that generates specific wavelengths.

A

spectrophotometer

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12
Q

The light path ___ through the cuvette, is ______ by the material in the cuvette, and is read by a ________.

A

passes; absorbed; detector

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13
Q

In the Bradford assay, the peak absorbance of ______ Coomassie G-250 dye is at ____ nm, and the _______ is set to read 595 nm.

A

unprotonated; 595 nm; spectrophotometer

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14
Q

___________ use standard curves created by measuring the ____________ of solutions of known concentration to determine the concentration of unknown samples.

A

Colorimetric assays; absorbances

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15
Q

What are 3 colorimetric methods for determining the total protein content of a sample?

A

biuret; Lowry and Bradford

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16
Q

Which is the oldest method and is commonly used in high school labs to detect the presence of a protein?

A

biuret

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17
Q

How many reactions does the biuret involve?

A

2: chelation and redox

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18
Q

Of the 3 colorimetric methods, which is the least sensitive?

A

biuret

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19
Q

Which of the 3 colorimetric methods involve 2 redox reactions?

A

Lowry

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20
Q

What is a redox reaction?

A

a type of chemical reaction that involves a transfer of electrons between two species.

Think the mnemonic OIL RIG.

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21
Q

What is OIL RIG?

A

Oxidation is the LOSS of electrons.

Reduction is the GAIN of electrons.

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22
Q

The formation or presence of bonds (or other attractive interactions) between two or more separate binding sites within the same ligand and a single central atom.

A

chelation

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23
Q

The Lowry assay is affected by ______ from many common lab reagents and chemicals.

A

interference

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24
Q

Of the 3 colorimetric methods, which is the most sensitive?

A

Bradford protein assay

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25
Q

The Bradford assay uses a dye, _____________, which was first described by M. Bradford in 1976.

A

Coomassie Brilliant Blue G-250

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26
Q

The Bradford protein assay takes advantage of the _____ properties of the dye and the dyes ability to interact with the side chains, or R-groups, of specific _______.

A

chemical; amino acids

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27
Q

As part of the Bradford solution, the dye exists in its _____ state and takes on a _________ color.

A

cationic; reddish-brown

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28
Q

The peak absorption of the Coomassie Brilliant Blue G-250 dye in cationic state is _____.

A

470 nm

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29
Q

When the Coomassie Brilliant Blue G-250 dye binds to and interacts with _______, the dye is converted to a stable _____ _____ form, and the absorption maximum shifts from 470 nm to _____.

A

amino acids; unprotonated blue; 595 nm

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30
Q

The stable blue form of the Coomassie Brilliant Blue G-250 dye is easily observed and quantified in a ________.

A

spectrophotometer

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31
Q

True or False? There is a correlation to the amount of blue color and the amount of protein in the sample.

A

True

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32
Q

The more protein, the more ____ blue color.

A

intense

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33
Q

By using a ______ series of known proteins, one can generate a spectrophotometric _____.

A

dilution; standard curve

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34
Q

The curve can be used to estimate the quantity of ___ in an unknown sample, based upon the ____ of ____.

A

protein; intensity; blue

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35
Q

The __________ is simple, highly _____, and relatively ________ by many common lab reagents and chemicals.

A

Bradford assay; sensitive; unaffected

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36
Q

How many types of interactions does the Coomassie G-250 dye bind to proteins?

A

3

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37
Q

What are the three types of interactions does the Coomassie G-250 dye bind to proteins?

A

1- Interaction through arginine (electrostatic bonding);
2- Interaction of aromatic rings of Coomassie (pi-electron stacking);
3- Interaction with polar amino acids that have hydrophobic R-groups (hydrophobic interactions)

38
Q

The primary interaction of the Coomassie G-250 dye with proteins occurs through ______, a very basic ________, which interacts with the _____ charged sulfate groups through _________ interactions.

A

arginine; amino acid; negatively; electrostatic

39
Q

Other weaker dye-protein interactions include the interaction of the __________ of Coomassie.

A

aromatic rings

40
Q

G-250 dye with the aromatic rings of amino acids, such as ________, through __________ interactions.

A

trytophan; electron stacking

41
Q

Finally, the G-250 dye also weakly interacts with ______ that have _____ R-groups, such as the aromatic ring of _____.

A

polar amino; hydrophobic; tyrosine

42
Q

The binding of the protein to the G-250 dye converts the dye to a ______, _______, _____ form.

A

stable; unprotonated; blue

43
Q

The ____ of the ____ color indicates the level of protein in a sample.

A

intensity; blue

44
Q

The more ____ the ____ color, the more _____ present in the sample.

A

intense; blue; protein

45
Q

How many main steps are there to perform the Bradford assay?

A

4

46
Q

The first step to perform the Bradford assay is:

Preparation of a _____ series or serial ____ of known ____ standards and preparations of unknowns.

A

dilution; dilutions; protein

47
Q

The second step to perform the Bradford assay is:

Addition of ______ dye (brown, ____ form) and _____ for >5 minutes (not to exceed ___ minutes).

A

Bradford; cationic; incubation; 60

48
Q

the introduction of microorganisms to growing media or substrates

A

inoculation

49
Q

allowing microorganisms to grow under supplied growth conditions

A

incubation

50
Q

The third step to perform the Bradford assay is:

Binding of dye to ____, resulting in color change to the blue ________ dye form and quantitative reading of the _________ at A595 in a ________.

A

protein; unprotonated; absorption; spectrophotometer

51
Q

The last and fourth step to perform the Bradford assay is:

Compilation of the data into a _____ and unknown protein ______ determination.

A

standard curve; concentration

52
Q

1 nm = ___ m

A

10^-6

53
Q

1 m = ___ nm

A

10^6

54
Q

What is the name of today’s lab?

A

Spectrophotometric determination of protein concentrations

55
Q

Which 3 amino acids are involved in electrostatic bonding in the Coomassie G-250 dye?

A

Arginine, lysine, histidine

56
Q

Which amino acids and components are involved in the hydrophobic interaction in Coomassie G-250 dye?

A

Leucine, other nonpolar amino acids, peptide backbone

57
Q

Which 3 amino acids are involved in the pi-electron stacking bonding in the Coomassie G-250 dye?

A

Tyrosine, tryptophan, phenylalanine

58
Q

In the experimental portion of this lab, what will be determined?

A

protein concentration of fractions

59
Q

In step 1 of the experiment, protein standards will be made. What will be measured against the standard concentration?

A

Absorbance

60
Q

What are the units of the standard concentration of the protein?

A

ug/ul

61
Q

In step 3, what is the protein-dye reagent that will be used?

A

Bradford Reagent

62
Q

What type of buffer will be used to dilute the samples?

A

Laemmli buffer

63
Q

What type of buffer will be used to unfold the proteins in the sample?

A

Laemmli buffer

64
Q

What type of buffer will add an overall negative charge?

A

Laemmli buffer

65
Q

Laemmli buffer is a mixture of four or more _____ and ____.

A

chemicals; additives

66
Q

The Laemmli buffers used here contains 4 components. What are they?

A

Bromophenol Blue;
Glycerol;
SDS;
BME

67
Q

What is the Bromophenol Blue used for?

A

tracking dye

68
Q

What is the Glycerol used for?

A

sinks the sample to the bottom of the well

69
Q

What is SDS used for?

A

helps denature the protein and add an overall negative charge

70
Q

What is BME used for?

A

to keep sulfide bonds from reforming

71
Q

What term is used to describe the unfolding of proteins?

A

Denature

72
Q

When the fractions have been evaluated for _______, they need to be prepared for _____.

A

protein concentration; SDS-PAGE

73
Q

At the end, the fractions need to be diluted 50/50 with _________ and heated at ____ for ___. They they must be stored in the _____.

A

Laemmli buffer; 95C; 5 min; freezer

74
Q

What is the subtitle of the experimental portion?

A

Determine Proteins Concentrations of Fractions

75
Q

In the electromagnetic spectrum, which type of wavelength has the MOST ENERGY?

A

Gamma Rays because it has the smallest wavelength.

76
Q

In the electromagnetic spectrum, which type of wavelength has the LEAST ENERGY?

A

Radio waves (eg. AM) because it has the longest wavelength.

77
Q

In the electromagnetic spectrum, which type of wavelength is the SMALLEST?

A

Gamma Rays

78
Q

In the electromagnetic spectrum, which type of wavelength is the BIGGEST?

A

Radio waves (eg. AM)

79
Q

What is the wavelength of visible light?

A

380nm 700 nm

80
Q

Visible light is dispersed upon passage through a prism. The dispersion of visible light starting from the longest to shortest wavelength is ____.

A

ROY G BIV: red, orange, yellow, green, blue, indigo, violet

81
Q

Visible light is dispersed upon passage through a prism. The dispersion of visible light starting from the MOST to LEAST ENERGY is ____.

A

VIB G YOR: violet, indigo, blue, green, yellow, orange, red

82
Q

What term describes an analysis done to determine the presence of a substance and the amount of that substance?

A

assay

83
Q

A chemical test for proteins and polypeptides. It is based on the _____ reagent, a blue solution that turns violet upon contact with proteins, or any substance with peptide bonds

A

biuret test

84
Q

With respect to waves, high energy = high frequency = short wavelength. True or False? If false, what do you need to do to make the equation true?

A

It’s true.

85
Q

With respect to waves, low energy = low frequency = short wavelength. True or False? If false, what do you need to do to make the equation true?

A

False.

low energy = low frequency = large wavelength.

86
Q

With respect to waves, low energy = low frequency = large wavelength. True or False? If false, what do you need to do to make the equation true?

A

It’s true.

87
Q

With respect to waves, high energy = low frequency = large wavelength. True or False? If false, what do you need to do to make the equation true?

A

False.

high energy = high frequency = short wavelength.

88
Q

With respect to waves, low energy = high frequency = large wavelength. True or False? If false, what do you need to do to make the equation true?

A

False.

low energy = low frequency = large wavelength.

89
Q

In Bradford’s assay, the peak absorbance of the dye-protein complex is _____.

A

595 nm

90
Q

In Bradford’s assay, the peak absorption of the dye is ____.

A

470 nm

91
Q

The primary interaction of the G250 dye with the proteins occurs through arginine, a very basic amino acid, which interacts with the negatively charged sulfate groups through electrostatic interactions. True or False?

A

True

92
Q

The Bradford assay is easy to perform and involves 4 main steps: place them in the correct order:

1- Take the sample reading using a lab experiment;
2- Prepare a dilution series of standards;
3- Make a sample curve and determine unknown concentrations;
4- Addition of dye.

A

2 4 1 3

BIOC 3622 - Biochemistry Lab (8 decks)

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