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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

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

A

Radiation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Tissues and cells are composed of what 4 different biomolecules?

A

DNA, proteins, lipids, carbohydrates

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

In what 4 ways can light interact with biomolecules?

A

reflection, absorption, transmission, light scattering

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

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

A

Bradford assay

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

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

A

light energy; light; wavelength

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

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

A

color

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

A __________ has a light source that generates specific wavelengths.

A

spectrophotometer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

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

A

passes; absorbed; detector

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

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

A

biuret; Lowry and Bradford

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

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

A

biuret

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

How many reactions does the biuret involve?

A

2: chelation and redox

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

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

A

biuret

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Which of the 3 colorimetric methods involve 2 redox reactions?

A

Lowry

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

What is OIL RIG?

A

Oxidation is the LOSS of electrons.

Reduction is the GAIN of electrons.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
23
Q

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

A

interference

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
24
Q

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

A

Bradford protein assay

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
25
The Bradford assay uses a dye, _____________, which was first described by M. Bradford in 1976.
Coomassie Brilliant Blue G-250
26
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 _______.
chemical; amino acids
27
As part of the Bradford solution, the dye exists in its _____ state and takes on a _________ color.
cationic; reddish-brown
28
The peak absorption of the Coomassie Brilliant Blue G-250 dye in cationic state is _____.
470 nm
29
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 _____.
amino acids; unprotonated blue; 595 nm
30
The stable blue form of the Coomassie Brilliant Blue G-250 dye is easily observed and quantified in a ________.
spectrophotometer
31
True or False? There is a correlation to the amount of blue color and the amount of protein in the sample.
True
32
The more protein, the more ____ blue color.
intense
33
By using a ______ series of known proteins, one can generate a spectrophotometric _____.
dilution; standard curve
34
The curve can be used to estimate the quantity of ___ in an unknown sample, based upon the ____ of ____.
protein; intensity; blue
35
The __________ is simple, highly _____, and relatively ________ by many common lab reagents and chemicals.
Bradford assay; sensitive; unaffected
36
How many types of interactions does the Coomassie G-250 dye bind to proteins?
3
37
What are the three types of interactions does the Coomassie G-250 dye bind to proteins?
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
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.
arginine; amino acid; negatively; electrostatic
39
Other weaker dye-protein interactions include the interaction of the __________ of Coomassie.
aromatic rings
40
G-250 dye with the aromatic rings of amino acids, such as ________, through __________ interactions.
trytophan; electron stacking
41
Finally, the G-250 dye also weakly interacts with ______ that have _____ R-groups, such as the aromatic ring of _____.
polar amino; hydrophobic; tyrosine
42
The binding of the protein to the G-250 dye converts the dye to a ______, _______, _____ form.
stable; unprotonated; blue
43
The ____ of the ____ color indicates the level of protein in a sample.
intensity; blue
44
The more ____ the ____ color, the more _____ present in the sample.
intense; blue; protein
45
How many main steps are there to perform the Bradford assay?
4
46
The first step to perform the Bradford assay is: Preparation of a _____ series or serial ____ of known ____ standards and preparations of unknowns.
dilution; dilutions; protein
47
The second step to perform the Bradford assay is: Addition of ______ dye (brown, ____ form) and _____ for >5 minutes (not to exceed ___ minutes).
Bradford; cationic; incubation; 60
48
the introduction of microorganisms to growing media or substrates
inoculation
49
allowing microorganisms to grow under supplied growth conditions
incubation
50
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 ________.
protein; unprotonated; absorption; spectrophotometer
51
The last and fourth step to perform the Bradford assay is: Compilation of the data into a _____ and unknown protein ______ determination.
standard curve; concentration
52
1 nm = ___ m
10^-6
53
1 m = ___ nm
10^6
54
What is the name of today's lab?
Spectrophotometric determination of protein concentrations
55
Which 3 amino acids are involved in electrostatic bonding in the Coomassie G-250 dye?
Arginine, lysine, histidine
56
Which amino acids and components are involved in the hydrophobic interaction in Coomassie G-250 dye?
Leucine, other nonpolar amino acids, peptide backbone
57
Which 3 amino acids are involved in the pi-electron stacking bonding in the Coomassie G-250 dye?
Tyrosine, tryptophan, phenylalanine
58
In the experimental portion of this lab, what will be determined?
protein concentration of fractions
59
In step 1 of the experiment, protein standards will be made. What will be measured against the standard concentration?
Absorbance
60
What are the units of the standard concentration of the protein?
ug/ul
61
In step 3, what is the protein-dye reagent that will be used?
Bradford Reagent
62
What type of buffer will be used to dilute the samples?
Laemmli buffer
63
What type of buffer will be used to unfold the proteins in the sample?
Laemmli buffer
64
What type of buffer will add an overall negative charge?
Laemmli buffer
65
Laemmli buffer is a mixture of four or more _____ and ____.
chemicals; additives
66
The Laemmli buffers used here contains 4 components. What are they?
Bromophenol Blue; Glycerol; SDS; BME
67
What is the Bromophenol Blue used for?
tracking dye
68
What is the Glycerol used for?
sinks the sample to the bottom of the well
69
What is SDS used for?
helps denature the protein and add an overall negative charge
70
What is BME used for?
to keep sulfide bonds from reforming
71
What term is used to describe the unfolding of proteins?
Denature
72
When the fractions have been evaluated for _______, they need to be prepared for _____.
protein concentration; SDS-PAGE
73
At the end, the fractions need to be diluted 50/50 with _________ and heated at ____ for ___. They they must be stored in the _____.
Laemmli buffer; 95C; 5 min; freezer
74
What is the subtitle of the experimental portion?
Determine Proteins Concentrations of Fractions
75
In the electromagnetic spectrum, which type of wavelength has the MOST ENERGY?
Gamma Rays because it has the smallest wavelength.
76
In the electromagnetic spectrum, which type of wavelength has the LEAST ENERGY?
Radio waves (eg. AM) because it has the longest wavelength.
77
In the electromagnetic spectrum, which type of wavelength is the SMALLEST?
Gamma Rays
78
In the electromagnetic spectrum, which type of wavelength is the BIGGEST?
Radio waves (eg. AM)
79
What is the wavelength of visible light?
380nm 700 nm
80
Visible light is dispersed upon passage through a prism. The dispersion of visible light starting from the longest to shortest wavelength is ____.
ROY G BIV: red, orange, yellow, green, blue, indigo, violet
81
Visible light is dispersed upon passage through a prism. The dispersion of visible light starting from the MOST to LEAST ENERGY is ____.
VIB G YOR: violet, indigo, blue, green, yellow, orange, red
82
What term describes an analysis done to determine the presence of a substance and the amount of that substance?
assay
83
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
biuret test
84
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?
It's true.
85
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?
False. low energy = low frequency = large wavelength.
86
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?
It's true.
87
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?
False. high energy = high frequency = short wavelength.
88
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?
False. low energy = low frequency = large wavelength.
89
In Bradford's assay, the peak absorbance of the dye-protein complex is _____.
595 nm
90
In Bradford's assay, the peak absorption of the dye is ____.
470 nm
91
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?
True
92
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.
2 4 1 3