Term
| Spectroscopy vs. Spectrometry |
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Definition
Spectroscopy
Light, or visible radiation, can be resolved into its component wavelengths (as with a prism)
Study of radiation interacting with matter
Study of molecular structure and dynamics through absorption, emission, and scattering
Spectrometry
Measurement of this interaction
Produces spectra that are used for theoretical studies on the structure matter, or concentration of a solution
Qualitative and quantitative analysis |
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Term
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Definition
Electromagnetic
Visible, UV, infrared, fluorescence spectroscopy
Flame spectroscopy
X-ray spectroscopy (crystallography)
Nuclear magnetic resonance spectroscopy (NMR)
Circular dichroism
Non-electromagnetic
Ions (mass spectroscopy)
Electrons (electron spectroscopy)
Sound waves (acoustic spectroscopy) |
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Term
| Fluorescence Spectroscopy |
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Definition
Used for studying biological systems
Very sensitive, highly specific and not destructive to the sample
Fluorescent molecules are excited, and then emit energy in the form of light as they return to the ground state |
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Term
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Definition
Similar to UV-vis, but less precise, wavelength region ~1000-200,000 nm
Energy transitions due to changes in vibrational, rotational & kinetic energy
Qualitative analysis of functional groups (carbonyls, alcohols, aromatics) |
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Term
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Definition
All substances in solution absorb light of one wavelength and transmit light of other wavelengths
Absorbance is a characteristic of a substance, like melting point, boiling point, density and solubility
Because absorbance can be related to the amount of the substance in solution, absorbance can be used to quantitatively determine the amount of the substance in solution |
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Term
| What is the rainbow angle? |
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Definition
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Term
| Spectrophotometer Components |
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Definition
Light source
Tungsten lamp – visible region, ~400-1000 nm
Deuterium lamp – UV region, ~200-400 nm
Monochromator – selects particular wavelengths of light for measurement
Prism or a grating – disperses light, and a narrow exit slit selects only a small range of the dispersed spectrum
Sample compartment – holds cuvettes for light to pass through to the detector
Detector
Phototubes, photomultipliers, or photodiodes
Photovoltaic effect – on receiving light of sufficient energy, electrons are emitted, which can be measured in the form of current by sensitive electronic devices |
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Term
| What device can read multiple samples at once? |
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Definition
Plate Reader
Read 96, 384, or 1536 samples in small volumes (5-200 ml)
Detect fluorescence, absorbance, other attributes
Used in ELISA, drug screening, enzyme assays, DNA/protein concentrations |
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Term
| What color is the human eye most sensitive to? |
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Definition
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Term
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Definition
260nM
This is the wavelength of light that is absorbed by DNA. This value is used to determine that concentration of DNA in your sample according to the conversion factor below
A260 of 1.0 = 50 μg/ml |
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Term
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Definition
280nM
The absorbance generated at 280 nm is used in the ratio A260:A280, which determines the purity of the DNA
Samples are considered of adequate purity if A260:A280 >1.5 |
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Term
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Definition
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Term
| Absorbency and Transmittance |
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Definition
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Term
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Definition
| (#Try)(5500)+(#Tyr)(1490)+(#Cys)(125) |
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Term
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Definition
Amino acid analysis
Absorbance at 280 nm
Chromogenic/colorimetric assays are used to construct a standard curve from samples containing known amounts of a purified protein (BSA), as in the DC and Bradford Assays (Lab 2, Lab 6)
Advantages – reproducible, simple to perform, fast, inexpensive, very useful
Disadvantages – only an estimate of protein concentration, interference from suphydryl derivatives, detergents, carbohydrates, amines, nucleic acids, lipids, salts |
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Term
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Definition
Most reliable way to determine the concentration of a pure protein
Acid hydrolysis of the protein
Separation of the amino acids
Determination of amino acid quantity
Determination of unknown proteins in sample
Protein purity essential
Special facilities, 2 days, and $40/sample
Unrealistic for routine determination of protein concentration |
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Term
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Definition
Quartz or UV compatible cuvettes must be used, as the basic plastic cuvettes will not transmit light at 280 nm
Most proteins have an absorption maximum at 280 nm
Ringed amino acids (trp, tyr, but not phe)
Molar absorptivity will vary greatly – proteins lacking trp or tyr will have no absorbance
Sample must be pure, as other proteins and nucleic acids can alter the absorption |
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Term
Quartz or UV compatible cuvettes must be used, as the basic plastic cuvettes will not transmit light at 280 nm
Most proteins have an absorption maximum at 280 nm
Ringed amino acids (trp, tyr, but not phe)
Molar absorptivity will vary greatly – proteins lacking trp or tyr will have no absorbance
Sample must be pure, as other proteins and nucleic acids can alter the absorption |
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Definition
| Protein (mg/ml) = (1.55 x A280) – (0.76 x A260) |
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Term
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Definition
Colorimetric = measuring changes in color
The more protein there is in a sample, the more intense the blue color
A spectrophotometer can be used to measure the degree of “blue-ness”
Even though a sample is more “blue” (and measured at 595, 750, etc.), it is a reduction of other wavelengths that we measure |
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Term
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Definition
Uses a dye called Coomassie Brilliant Blue, which changes color from red (465) to blue (595) as the dye binds to proteins
Dye forms strong, noncovalent (van der Waals) complexes with proteins
Relative to number of + charges on a protein
Associated with amino acids (arginine, phenylalanine, trytophan, histidine, proline)
van der Waals forces with amino and carboxyl groups
Hydrophobic interactions
Does not detect free amino acids, or anything less than 3000 Da
Advantages
Sensitive, accurate, very fast
Compatible with most
Common buffers
Chaotropic reagents (6M guanidine-HCl, 8 M urea, sodium azide)
During protein purification, can be used as a quick “flow through” check
Disadvantages
Color response is nonlinear over a wide range of protein concentration, therefore a standard curve should be run with each assay
2X the variability as copper methods
Some proteins that are insoluble in the acidic dye cannot be assayed
Incompatible with surfactants, detergents
Reagent stains cuvettes
Linear range – 0.05 to 0.5/1.5 mg/ml depending on kit
38-45% protein to protein variation |
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Term
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Definition
In alkaline conditions, copper(II) binds to the peptide nitrogen of proteins
Cupric complex absorbs light at 550 nm
Since the copper reacts with the peptide bond, there is little interference by free amino acids, and the amino acid composition of the proteins is not very important
Not very sensitive
Buffer interference
tris, ammonia |
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Term
| Lowry Method (DC Protein Assay) |
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Definition
Commonly used method (inexpensive, easy, reproducible)
Method
Copper binds peptide bonds under basic conditions
Cu2+ Cu+
Cu+ reacts with the Folin reagent (phosphomolybdic-phosphotungstic reagent) which becomes reduced to blue (750 nm)
Standard curves are linear only at low protein concentration, therefore a standard curve is run with each assay
Timing/mixing of reagents with the samples must be precise, reagents unstable
Sensitive to contaminants
Detergents (Lowry, not DC), lipids, sugars, pH
Linear range – 0.2 to 1.5 mg/ml depending on kit |
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Term
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Definition
Similar to Lowry reaction using BCA (bicinchoninic acid) reagent instead of Folin, faster and easier, more stable reagents
Cu2+ is reduced to Cu+
Two molecules of BCA chelate to a copper ion
Sensitive to contaminants
Carbohydrates, catecholamines, tryptophan, lipids, phenol red, cysteine and tyrosine, impure sucrose glycerol, H2O2, uric acid, iron
Low protein to protein variation (15%)
Intense purple color
Max absorption at 560 nm |
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Term
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Definition
The absorbance values of the lysate do not give us any correlation between absorbance and protein concentration
This problem can be solved by performing a standard curve
By graphing the relationship between absorbance and the known concentration of BSA, a correlation can be made between absorbance and protein concentration |
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