experiment 5: determining total protein amount in rGFP fractions Flashcards
based upon the design and principle of spectrophotometry, what improper lab techniques could result in incorrect absorbance readings?
- SCRATCHING: rubbing the cuvettes too hard leaves microscopic scratches on the surface of the cuvette. this causes scattering of light, and therefore an inaccurate absorbance reading that leads to REFLECTION
- AIR BUBBLES/ BACTERIA TURBIDITY: leads to SCATTERING of light
- FINGERPRINTS/OIL: leads to ABSORPTION
given the conditions of an enzyme assay, describe what components of that assay should be placed in the reference cuvette to properly “blank” the machine
a “blank” solution contains everything accept the protein of interest. therefore, it is a 1:10 dilution. there should be a sample with a low level, almost non-existent level of reflection, or refraction. once the machine has a base line, it makes it easier to detect the absorbance in the sample.
define wavelength
the distance between two points in the same phase in consecutive cycles of a wave
define wavelength scan
measures the absorbance and emission of light through a sample. absorbance is proportional to concentration and a wavelength scan can be used to determine concentrations of a sample.
define maximum wavelength
“Maximum wavelength” is shorthand for “wavelength of maximum absorption” and since you’ve got wavelength on the x-axis and absorption on the y-axis, it’s just the wavelength at the very top of the largest peak. Lambda max is the wavelength at which the maximum fraction of light is absorbed by a solution.
define absorbance and transmittance
the absorbance of a material is a logarithmic ratio of the radiation falling upon a material to the radiation transmitted through a material while transmittance is the fraction of incident light at a specified wavelength that passes through a sample
what are the principle differences between a spectrophotometer and a spectrofluorometer?
a spectrophotometer measures absorbance while a spectrofluorometer measures fluorescence
define specific activity
specific activity is an estimate of purity where high specific activity correlates with high purity. it is calculated by dividing the activity of your protein of interest by the total amount of protein.
what is the procedure for quantifying protein concentration that we used in lab 5?
the binding of Coomassie dye to proteins causes a shift of absorption maximum from 465nm (red) to 595 (blue). dye forms noncovalent complexes with proteins via electrostatic interaction with primary amines and carboxyl groups via van der walls forces. this interaction results in a color shift from red/brown to dark blue. color response is nonlinear over a wide range of protein concentration. the dye is added to the solution to be measured and incubated at room temp. and then absorbance is measured at 595 nm.
what are the underlying principles of Biuret assay?
the Biuret assay measures the amount of nitrogen in a peptide bond. this is done by converting copper 2+ to copper 1+, which allows it to absorb more light at 550nm.
- advantages: amino acid composition of proteins is not important and little interference by free amino acids
- disadvantages: tris and ammonia interfere and cause increased absorbance values and the assay is insensitive
what are the underlying principles of a 260/280 assay?
this assay measures absorbance of a fraction at 280nm (UV light). proteins have absorption max at 280nm due to tryptophan, tyrosine and phenylalanine, and varies due to the amount of these in protein.
- advantages: does not destroy the sample, is very fast, really simple to do.
- disadvantages: only accurate if protein is pure and molar absorptivity is known, purine and pyrimidine rings have absorbance was at 260 but can also go as high as 280 meaning nucleic acids are hard to measure using this assay
what are the underlying principles of a Lowry assay?
the Lowry reaction consists of the Biuret reaction
followed by the reduction under alkaline conditions of the Folin—Ciocalteu reagent. copper ions facilitate the reduction process. the principal chromogenic groups are the peptide linkages in complex with copper and the blue-reduced molybdotungstates, which are largely reduced by Tyr, Trp, and polar amino acids. therefore, the sensitivity of the test depends on the composition of the protein. the product of the reaction, heteropolymolybdenum blue, is strongly blue with an absorption maximum of ~750 nm
- advantages: inexpensive, easy, very sensitive, and highly reproducible.
disadvantages: sensitive to variety of contaminants, standard curves are linear only at low protein concentrations, timing and mixing has to be precise, standard curve needs to be created each time protein determinations are done.
what are the underlying principles of a Bradford assay?
the bradford assay indicates the amount of certain proteins due to shifting absorbance from 465nm to 595nm in acidic solution by forming strong noncovalent complexes with proteins via interactions with amino and carboxyl groups via van Der Waals forces.
- advantages: sensitive and accurate, easy one step. compatible with most common buffers.
- disadvantages: reagent stains cuvettes and can be difficult to remove, high concentrations of detergents can interfere with this assay
