Lab 9 Protein Analysis Flashcards
(84 cards)
1
Q
Fish fraud is only an economical concern.
True or False?
A
False.
Cheaper fish species are not always safe for human consumption, so fish fraud is also a food safety concern.
For example, escolar, often sold as white tuna, can cause diarrhea-like symptoms, and amberjack often sold as yellowtail tuna, can have a naturally occurring toxin that causes neurological symptoms.
2
Q
What is ‘crude protein’?
A
Protein (muscle, myofibrillar, and stroma) and non-protein nitrogen (generally from sarcoplasm)
3
Q
What is PAGE?
A
Polyacrylamide gel electrophoresis
A technique in which proteins are separated in a cross-linked acrylamide gel matrix.
4
Q
How are proteins separated in SDS-PAGE?
A
- Proteins are denatured using heat, mercaptoethanol, and anionic detergent (SDS)
- Separation takes place on the basis of size, since the SDS makes all proteins uniformly negative in charge.
SDS = sodium dodecyl sulfate
5
Q
How are proteins separated in native-PAGE?
A
- Proteins are separated based on their native charge and molecular weight.
6
Q
How are proteins separated in IEF-PAGE?
A
- Native proteins are separated by native charge in a pH gradient.
- Each protein migrates until it reaches the pH of its isoelectric point (the point where there is no net charge on the protein).
- Proteins with a net positive charge (below its isoelectric point) will migrate toward the cathode.
- Proteins with a net negative charge (above its isoelectric point) will migrate toward the anode.
IEF = isoelectric focusing
7
Q
In IEF-PAGE proteins with a net positive charge (below its isoelectric point) will migrate toward the cathode.
True or False?
A
True.
8
Q
In IEF-PAGE proteins with a net positive charge (below its isoelectric point) will migrate toward the anode.
True or False?
A
False.
In IEF-PAGE proteins with a net positive charge (below its isoelectric point) will migrate toward the cathode.
9
Q
In IEF-PAGE proteins with a net negative charge (above its isoelectric point) will migrate toward the anode.
True or False?
A
True.
10
Q
In IEF-PAGE proteins with a net negative charge (above its isoelectric point) will migrate toward the cathode.
True or False?
A
False.
In IEF-PAGE proteins with a net negative charge (above its isoelectric point) will migrate toward the anode.
11
Q
Which type of PAGE is used to authenticate food samples?
How does it work?
A
IEF-PAGE
- The species from which a food sample was obtained can be determined by matching the pattern arising from the protein bands with known standards.
- Proteins from different species will have a unique pattern when run on an IEF-PAGE because of different amino acid compositions, even if the molecular weight of the proteins from the two different species are almost identical.
12
Q
You want to conduct a milk protein analysis using a casein protein standard curve. Your standard curve will have 8 points and 3 technical replicates and you will also prepare 3 technical replicates of 2 diluted milk samples. 200 µL of BCA reagent will be added to each sample and standard.
What is the minimum volume of BCA reagent you should prepare?
A
6.6 mL
13
Q
What wavelength do you use when carrying out a BCA assay?
A
562 nm
14
Q
What is the principle behind the Bicinchoninic Acid Method protein assay?
A
Reduction of Cu2+ to Cu1+ by protein in an alkaline solution and the ions will be trapped by two molecules of BCA to create a purple-colored product
15
Q
What is the problem when your protein standard curve is constructed using concentrations 0 – 2.0 mg/ml (with absorbance of 0.134 – 0.843) but the absorbance of your sample is 0.953?
A
You cannot ensure that the relationship between concentration and absorbance is still linear to extrapolate the result
16
Q
What colour should your BCA working reagent be?
A
Green
17
Q
In this example:
How much of each standard concentration do you need?
How much of the fish sample do you need?
How much total BCA reagent do you need?
A
Standard concentration: 10 uL x 3 = 30 uL
Sample: 10 uL x 3 = 30 uL
Reagent: (6 standards + 1 sample) x 200 uL x 3 replicates = 4200 uL
18
Q
Fill in the table – choose 6 equally spaced dilutions between 0 and 2 mg/mL including 0 mg/mL as one of the 6 standards. Prepare 1 mL of each standard
2.0 mg/mL standard
A
I
19
Q
Now, let’s prepare 2 dilutions of the fish sample:
* Fish are known to contain between 20 – 30 g protein/100 g of fish
* The protein extraction method requires us to homogenize 25 g of fish in
100 mL of water
* What is the possible protein concentration range in this homogenous solution?
A
20
Q
A
21
Q
What is the mechanism of the BCA assay?
A
- Peptide bonds in protein reduce Cu2+ to Cu1+ in the presence of a base
- BCA reagent is alkaline (~pH 11)
- Temperature-dependent assay!!
- Every assay needs a new standard curve
- The amount of Cu2+ reduced is proportional to the amount of protein in the sample
- BCA reagent chelates the Cu1+ from the protein to form a complex
- 2 moles of BCA to 1 mole of Cu1+
- Not a reaction, no bonds are formed
- Creates a colored product that can be quantified at λ = 562 nm
22
Q
The BCA assay involves a reaction between the BCA reagent and copper.
True or False?
A
False.
No bonds formed.
BCA reagent chelates the Cu+1 (reduced from Cu2+ by peptide bonds in protein) to form a complex
23
Q
The BCA assay is temperature-dependent.
True or False?
A
True.
Every assay needs a new standard curve.
24
Q
The BCA agent is acidic.
True or False?
A
False.
It is alkaline.
25
The BCA reagent is alkaline.
True or False?
True.
26
You discovered the casein solution you used to construct a standard curve was actually 1.7 mg/mL, not 2 mg/mL. How will this affect the protein concentration you determine?
Overestimation
27
What happens if the reagent changes from green to purple before adding a protein sample?
## Footnote
Biuret reaction a.k.a. BCA protein assay
Glassware or pipette tips contaminated reagent (very sensitive to metal ions)
28
How do you prepare BCA reagent from reagents A and B?
50 parts A to 1 part B
29
How long is prepared BCA working reagent stable?
For a short duration in a closed container at room temperature
30
Describe the basis of the Kjeldhal method of protein determination.
* Determination of the % total nitrogen in a sample
* Convert to % protein using a conversion factor that varies with different food matrices.
31
What are the three basic steps of the Kjeldahl method?
1. Digestion
2. Neutralization and distillation
3. Titration
32
What happens during the digestion step of the Kjeldahl method?
* Proteins and other organic food components are digested by sulfuric acid in the presence of catalysts.
* Protein nitrogen is liberated to form ammonium ions
* Carbon and hydrogen elements are converted to carbon dioxide and water.
33
What happens during the neutralization and distillation step of the Kjeldahl method?
* Sulfuric acid is neutralized with alkali
* The ammonium is distilled into an acid solution
34
What happens during the titration step of the Kjeldahl method?
* The % ammonium (or % N) in the solution is determined via back-titration
* This value is converted to % protein using the conversion factor that depends on the analyte
35
Why does the Kjeldahl method determine 'crude protein' content only?
* Nitrogen also comes from nonprotein components (e.g., ammonia and ammonium sulfate)
36
What is the principle by which proteins are separated using SDS-PAGE?
Molecular size
37
What is the principle by which proteins are separated using IEF-PAGE?
isoelectric point
38
What form of acrylamide is toxic to humans?
Unpolymerized
39
Which solution should be added last when preparing the resolving and stacking gels?
TEMED
40
What is used to flatten down the resolving gel to produce an evenly distributed gel matrix?
Isopropanol
41
What is the purpose of digestion in the Kjeldahl method?
Release ammonium ions from proteins to be indirectly quantified
42
What is the purpose of distillation in the Kjeldahl method?
To convert ammonium ions to ammonia gas
43
What is the purpose of titration in the Kjeldahl method?
Directly quantify total moles of HCl remaining
44
The wells in the SDS-gel should be filled with running buffer before the samples and standard are added.
True or False?
True.
45
The wells in the SDS-gel should be filled with running buffer after the samples and standard are added.
True or False?
False.
The wells in the SDS-gel should be filled with running buffer before the samples and standard are added.
46
What should you do after you have run your SDS gel and removed the glass plates?
Remove the stacking gel
47
What is the difference between stacking gel and resolving gel?
* The acrylamide concentration determines the different pore sizes of the polyacrylamide gel. The stacking gel has a lower acrylamide concentration (5%) so the pore size is larger. When an electrical charge is applied to the gel, the proteins move through this part of the gel at a similar speed.
* The resolving gel has a higher acrylamide concentration (usually 10 to 15%), meaning it has a smaller pore size than the stacking gel. This part of the gel separates the proteins by their molecular weight. Larger proteins—which have a higher molecular weight—move through the pores in the resolving gel slower than smaller proteins—which have a lower molecular weight.
48
What is Laemmli buffer and what is it used for? [3]
* Sample loading buffer or sample dissociation buffer
* It contains (1) dye to assist in the monitoring of movement when running a gel.
* It also contains (2) SDS, which helps mobilize the proteins in the gel
* Lastly, (3) the beta-mercaptoethanol, upon mixing with the sample and heating, reduces disulfide bonds. This reduces multimeric protein down to their constituent proteins
49
What is a running buffer?
* Running buffer contains ions that conduct the current being applied through the gel, in turn allowing the proteins (which are covered with SDS, which is charged) to move through the gel.
50
What is the purpose of the Kjeldahl tablets?
* The metals in the chemicals act as catalysts.
* The K2SO4 (and also sulfate from CuSO4) aids in elevating the boiling point, which allows the liquid phase to get over 100 degrees C and thereby increase the reaction rate/extent.
51
What is the benefit of GelCode blue/Coomassie stains? [2]
* They bind all proteins
* The staining protocol is streamlined
52
Describe 4 applications of protein electrophoresis.
* Protein expression
* Molecular weight (MW) of proteins
* Presence of certain proteins (via Western blotting with antibodies)
* Isolate proteins for mass spectral analysis (cut band of interest out, digest gel, and isolate protein again)
53
Polyacrylamide gels are used to separate proteins.
What is the gel made of?
Acrylamide (A) and *bis*-acrylamide (B)
54
Polyacrylamide gels are used to separate proteins.
What initiates gel formation?
A free radical (APS) and a free radical stabilizer (TEMED)
## Footnote
APS (ammonium persulfate) and TEMED (tetramethylethylenediamine) are used to initiate the polymerization of acrylamide and a comonomer crosslinker such as bis-acrylamide to form polyacrylamide gels. APS acts as an initiator by decomposing to form free radicals that can initiate polymerization. TEMED acts as a catalyst by accelerating the rate of polymerization.
55
Polyacrylamide gels are porous - which allows for proteins to move through.
What defines pore size?
* % acrylamide (as % increases, pore size decreases)
* % *bis*-acrylamide (cross-linker) (as % increases, pore size decreases; minimum pore size occurs at 5% *bis*-acrylamide)
56
What does the pore uniformity of polyacrylamide gel depend on?
* The amount of catalyst (free radical) used
* Ammonium persulfate (APS) - free radical source
* Tetramethylethylenediamine (TEMED) - helps stabilize the free radicals from the persulfate
57
What is sodium dodecyl sulfate (SDS) used for in protein electrophoresis?
* Denatures proteins
* Binds them via hydrophobic interactions and imparts a uniform negative charge
58
What is 2-mercaptoethanol used for in protein electrophoresis?
* Reduces disulfide bonds allowing for full denaturation/unfolding
59
Describe SDS-PAGE sample preparation.
* Protein is uniformly covered in negative charge (~1 SDS per 2 amino acids)
* SDS blocks any positive charges on the protein
* Regardless of the size of the protein, they all have the same charge:mass ratio
60
Describe the SDS-PAGE set-up.
* Cathode (-) at the top
* Anode (+) at the bottom
61
Describe protein separation via SDS-PAGE.
* Movement of negatively charged proteins through gel pores is due to an applied electric current across the gel.
* Rate of movement is inversely proportional to the molecular weight
* Bigger protein = less movement
* Smaller protein = more movement
* Remember: same charge: mass ratio
62
What is a stacking gel?
* Low % acrylamide = larger pore size
* Does not restrict protein movement by size
* Used to align proteins at interphase of two gels in order to achieve accurate separation
63
What is a resolving gel?
* Higher % acrylamide = smaller pore size
* Used for size separation of proteins
64
Describe how the different gels work together in SDS-PAGE.
65
Describe coomassie brilliant blue dye.
* Binds to proteins through ionic interactions (sulfonic groups on dye and amine groups on protein)
* General van der waals interactions
* Can detect as little as 0.1 ug of protein
66
Describe pros and cons of silver staining.
* Pro: can detect as little as 1 ng of protein (100x more sensitive than Coomassie)
* Con: lengthy procedure, reagents used interfere with downstream analyses such as mass spectrometry)
67
What is a protein ladder?
* Standards of known molecular weight must be run on every gel
* Movement of ladder standards will vary with gel preparation and electrophoresis conditions.
68
Given a protein ladder, how may protein molecular weight be determined?
* Extract relative migration of standards and plot log(MW) versus. migration
* Standard curve then used to determine molecular weight of unknown proteins or to identify proteins
69
How does IEF-PAGE work?
* separation based on the isoelectric point (pl) of a protein)
70
Describe protein separation via IEF-PAGE.
* Gel is a lower % polyacrylamide gel; does not restrain the movement of proteins by size
* Uses a pH gradient gel
* Above its pI, a protein is negatively charged and will migrate towards the anode (+)
* Below its pI, a protein is positively charged and will migrate towards the cathode (-)
* Proteins migrate until they reach their pI’s
* No net charge and therefore does not move further
71
How is the pH gradient formed in IEF-PAGE?
* Established using ampholytes, that is: small molecular weight molecules that possess both acidic and basic functional groups and form pH gradients under the influence of electric fields
72
What is the equivalent of a protein ladder for IEF-PAGE pI determination?
* Standards of known pI must be run on every gel
* Results reported as relative distance travelled from cathode
73
Summarize Kjeldhal Protein Determination.
**1) Digestion**
* Proteins and other organic materials are broken down by sulfuric acid
* Protein nitrogen is released in the form of ammonium ions (NH4+)
**2) Neutralization and distillation**
* NaOH is added to convert NH4+ to NH3 (gas)
* NH3 is distilled (separated) into an acid solution (HCl)
**3) Titration**
* NH3 neutralizes some of the HCl
* Remaining HCl is then back-titrated (with NaOH) to determine how much ammonium was present
* %N is then determined and converted to % protein by a conversion factor (food specific)
74
What is the purpose of mercaptoethanol in SDS-PAGE?
In SDS-PAGE, mercaptoethanol is used as a reducing agent to break disulfide bonds which can form between cysteine amino acids in some proteins. This reduction of disulfide bonds is important for allowing the protein to become completely unfolded so that it migrates properly for its molecular weight.
75
What is the purpose of the anionic detergent in SDS-PAGE?
Anionic detergents such as SDS (sodium dodecyl sulfate) are used in SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) for protein solubilization, linearization and for establishing a uniform charge in preparation for gel electrophoresis. SDS binding denatures the polypeptides and imparts a negative charge that masks their intrinsic charge.
76
Why does detection of fish fraud generally not use SDS-PAGE?
- Because if the MW of proteins of some species may be almost identical and hard to differentiate.
- IEF-PAGE can discern between them because of different amino acid compositions
77
The BCA assay is more precise than the Kjeldahl method.
True or False?
False.
The Kjeldahl method is more precise.
78
The BCA assay is less precise than the Kjeldahl method.
True or False?
True.
The Kjeldahl method is more precise.
79
What is the purpose of the TRIS buffer in preparation of gels for SDS-PAGE?
Tris buffer is used in the preparation of SDS-PAGE gels to maintain a stable pH during the electrophoresis process. The pH of the buffer affects the overall charge of the proteins being separated, which in turn affects their migration through the gel. Tris buffer is commonly used because it has a high buffering capacity and can maintain a stable pH over a wide range of temperatures. This helps to ensure consistent and reproducible results in SDS-PAGE experiments.
80
Why is the BCA assay less precise than the Kjeldahl method?
* BCA assay can be affected by interfering substances, variations in protein composition, and temperature. It is also an indirect method that relies on reduction of copper ions by protein in an alkaline solution.
* The Kjeldahl method is a direct measurement of nitrogen content.
81
Describe the purpose of the components of the Laemmli buffer. [4]
* **SDS** (sodium dodecyl sulfate): a detergent that denatures the proteins and gives them a uniform negative charge, allowing them to be separated based on their size.
* Reducing agents (**β-mercaptoethanol**): these break disulfide bonds within and between protein molecules, further denaturing the proteins.
* **Glycerol**: increases the density of the sample, allowing it to sink to the bottom of the well when loaded onto the gel.
* **Bromophenol blue**: a tracking dye that allows you to monitor the progress of the electrophoresis.
82
What is the 20% TCA solution for in IEF-PAGE? What about the 30% MeOH:10% acetic acid:60% ddH2O solution?
The fixing solution (20% TCA) is used after the electrophoresis step to fix the proteins in place within the gel. This is done by crosslinking the proteins to the gel matrix, preventing them from diffusing out of the gel during subsequent steps. The fixing solution typically contains a mixture of alcohols (such as methanol or ethanol) and an acid (such as acetic acid) that help to dehydrate and shrink the gel, further immobilizing the proteins.
The washing solution (30% MeOH: 10% acetic acid: 60% DDH2O) is used after the fixing step to remove any excess fixing solution and unbound proteins or other contaminants from the gel. This helps to improve the clarity and resolution of the final stained gel. The washing solution typically contains water or a mild buffer to gently rinse the gel without disrupting the fixed proteins.
In summary, the fixing solution is used to immobilize the proteins within the gel, while the washing solution is used to remove any excess reagents and contaminants from the gel.
83
What is the end point in a Kjeldahl titration?
Solution changes from pink to orange
84
What is the role of mercaptoethanol in lysine determination in milk?
OPA (o-phthaldialdehyde) reacts with primary amino groups in the presence of 2-ME to form a highly fluorescent isoindole derivative. The 2-ME reduces the OPA molecule, allowing it to react with the primary amino group to form the fluorescent product.
