Exam I Flashcards
What is the range of a P10 micropipette?
.2 microliters to 10 microliters
What is the range of a P-20 micropipette?
2 microliters to 20 microliters
What is the range of a P200 micropippette?
20 microliters to 200 microliters
What is the range of a P1000 micropippete?
200 microliters to 1000 microliters
What are important considerations for micropipettes?
Never
- over-rotate
- use without a tip in place
- lay the pipetteman down when it contains liquid
- let the plunger snap back
- immerse the barrel of the pipetman
Always use a disposable tip
How can you express concentration?
Mass % = mass of solute/volume of solvent
Parts per million = 1g/1000000 mL
Molarity (M) - moles of solute/L of solvent (used most often in this class)
Morality (m) - moles of solute/ kg of solvent
What are the three variations of expression consentration?
Weight per volume %
Volume per Volume %
Weight per Weight %
How is molecular weight expressed?
G/mol
How is the molecular weight of a protein expressed?
Daltons = g/mol
What is the equation to calculate the amount of solid to weigh out to make a given volume of solution at a particular concentration?
G/mol * mol/L * L = g
MW of solid * required molarity * required volume = amount to be weighed
How do you convert from % solution to molarity?
Molarity = (% solution * 10)/MW
What does percent mean?
Parts per hundred grams or hundred ml
What is the general formula to convert dilutions?
C1V1 = C2V2
What involves techniques to monitor the interaction of light with matter?
Spectrophotometer
What is a form of electromagnetic radiation that has two components: electric and magnetic, which oscillate perpendicular to each other?
Light
What equation gives the energy of electromagnetic radiation (EM)?
E = hv
V is frequency, Hz, 1/sec
H is planck’s constant = 6.62410^-34 Jsec
What equation gives frequency?
V=c/lambda
C = speed of light (2.998*10^8 m/s)
Lambda is the wavelength
What is the range of wavelengths for visible light?
340 to 800 no
What is the range of wavelengths for UV light?
200 to 340 no
In what state do the electrons spin opposed to each other?
Singlet
In what state do electrons spin parallel to each other?
Triplet
Which state has the lower energy?
Triplet
Singlet to triplet transition has a very low what?
Probability
What transition of state to state is more probable?
Singlet to singlet
When energy goes from ground state to a singlet we say that the light was what?
Absorped
When energy goes from a singlet to ground state we call it what?
Fluorescence
When energy goes from a triplet to ground state we call it what?
Phosphorescence
When wavelengths are absorbed the transmitted wavelengths will be seen as what?
Complementary color
What is a plot of the amount of light absorbed by a sample as a function of wavelength?
Absorption spectrum
What molecules go from sigma to anti bonding sigma?
Aliases
What molecules go from pi bonds to anti bonding pi bonds?
Alkenes
Carbonyl compn
Sulfur
Halogen compounds
What molecules go from non bonding to anti sigma bonding?
Oxygen, nitrogen, sulfur, and halogen compounds
What molecules go from non bonding to anti pi bonds?
Carbonyl compounds
What is a weak transition in the range of 200 to 800 nm?
Non bonding to anti pi bonds
What is an intense transition in the range of 200 to 800 nm?
Pi to anti pi bonds
Increasing the number of conjugate does what?
Increase the wavelength absorbed
What is a covalently unsaturated group responsible for electronic absorption?
Chromosphore
What is a saturated group with non bonded electron which when attached to a chromophore alters both the wavelength and intensity of the absorption?
Auxochrome
What is an increase in absorption intensity?
Hyperchromic shift
What is a decrease in absorption intensity?
Hypochromic shift
What is a shift in absorption to a longer wavelength due to a substitution or solvent effect
Red shift (Bathochromic shift)
What is a shift in absorption to a shorter wavelength due to substitution or solvent effects?
Hypsochromic shift (blue shift)
What equation gives absorbance?
A = 2 - log %T
What is the Beer-Lamber law?
A = molar absorptivity (extinction coefficient) * concentration (M) * path length (cm)
Does Absorbance have units?
NO
What equation gives %T using the Beer-Lambert’s Law?
%T = 10^(2 - epsilon * c * l)
What is lost at high concentration?
Linearity
What is constant for a particular substance at a given wavelength?
Molar absorptivity or extinction coefficient
What equation can be used if the sample is the same but measured at the same wavelength? ie to find a concentration.
A1 c2 = A2 C1
A1/c1 = A2/c2
What are some applications of UV-Vis spectrophotometry?
Determine chromophore composition
Cell density
Determination of concentration
Measure structural changes/reaction - example - enzyme assays
What are two other ways the extinction coefficient can be expressed?
E% = (Emolar *10)/MW
Emg/ml = Emolar/MW
What can happen to a molecule at different conditions ie pH change?
Structural changes that effect absorbance and wavelength.
Shifts in absorbance and wavelength can be used to monitor what?
Reactions
When measuring turbidity or cell density, what is measured?
The scattering of light
Proteins can absorb at what two wavelengths?
220 nm peptide bonds
280 nm aromatic side chains
Why do we usually only use the 280 nm wavelength?
Too much background absorbance at 220 nm
Spectrophotometer is preferred to analyze proteins because of what?
The sample is recoverable
What is the part of a molecule that absorbs light from the UV-vis part of the spectrum called?
Chromophore
N->pi* is weak or intense?
Weak
Pi->pi* is weak or intense?
Intense
What is the Ligand-Field Theory?
D-block elements/transition state metals also absorb light in the visible range
Absorption shifts where the wavelength increases are called what?
Red shifts or bathochromic shifts
Absorption shifts that decrease the wavelength are called what?
Blue shifts or hypsochromic shift
Increases in absorbance are called what?
Hyperchromic shift
Decreases in absorbance are called what?
Hypochromic shifts
With increasing aromatic rings the length of the wavelength does what?
Increase
Which Amino acid residues have the ability to absorb at 280 nm?
Tryptophan, Tyrosine, Cystine (a dimer of cysteine)
Is cytosol reducing or oxidizing?
Reducing
Is air reducing or oxidizing?
Oxidizing
What measurement is used to determine the optical density of cells?
600 nm
Why does DNA have a higher UV absorption compared to proteins?
The presence of aromatic rings
Why does DNA absorption increase as it melts?
Non bonded electrons interact with pi bonding
List some limitations of the Beer-Lambert law
Deviations in epsilon at high concentration due to electrostatic interactions between molecules, changes in refractive index, and shifts in the chemical equilibria
Scattering of light due to particulates in the sample
Fluorescence or phosphorescence of the sample
Non monochromatic radiation
Stray light
What is used to indirectly detect protein concentrations?
Either copper chelation chemistry
Or
Binding to dye
What are the three methods that use copper chelation chemistry
Biuret method
Lowry Assay (Folin-Cicalteu reagent)
BCA method
What is the name of the method to determine protein concentration through binding to dye?
Bradford Assay (coomassie Blue)
How does the Biuret Method work?
Cu2+ binds to protein and gets reduced to Cu+ which forms a Biuret complex that absorbs light at 550 nm (blue-purple)emitted
What are the advantages of the Biuret Method?
Applicable to any protein irrespective of the presence of aromatic amino acids
What are the disadvantages of the Biuret Method?
Not very sensitive (requires a large amount of protein)
How is the Lowry Assay different from the Biuret Method?
A phosphomolybdic-phosphotungstinic acid solution is used. This allows tyrosine, tryptophan, cysteine, histidine, and asparagine to enhance the amount of color used (the contribute additional reduction fo the phosphomolybdic/phosphotungstinic acid complex)
What are the advantages of the Lowry Assay?
100 fold more sensitive than Biuret
What are the disadvantages of the Lowry Assay?
Absorbance depends on the composition of the protein which makes a protein stand hard to find
Many substances can interfere (Tris buffer, ammonium sulfate, EDTA, reducing agents,
What wavelength is absorption found at for the Lowry Assay?
750 nm
How is BSA different than the Biuret method?
Bichinochonic acid is added to the solution
At what wavelength does absorption occur when using the BCA method?
562 nm
What are the advantages of the BCA method?
Colored complex is stable (100 times more sensitive than biuret method)
What are the disadvantages of the BCA Method?
Bicinchonic acid is expensive
Composition is dependent on cysteine/cystine, tyrosine and tryptophan (increase the color)
What is the process of the Bradford Assay?
An acidic solution of Coomassie Brilliant Blue G-250 is used
At what wavelength does unstable (cationic) CBB absorb?
465 nm
At what wavelength does stable (anionic) CBB absorb at?
595 nm
What are the advantages of the Bradford Assay?
Very sensitive, fast, inexpensive, compatible with a wide range of substances, dye-protein complex is stable, but requires a standard bovine albumin serum (BSA)
What do you plot to generate a standard curve when using the Bradford Assay?
Absorbance vs concentration
What is the minimum Rsquared value that is acceptable when generating a standard curve?
0.98
What is the shift in wavelengths called when comparing the absorbance spectrum with the fluorescence emission spectrum called?
Stokes shift
What is the Forster Resonance Energy Transfer (FRET)?
When one molecule absorbs at a shorter wavelength and emits at a longer wavelength which is then absorbed by a second molecule which may or may not emit at an even longer wavelength.
According to Svante Arrhenius, what is an acid and a base?
An acid is a material that can release a proton and a base is a material that can donate a hydroxide ion.
According to Lowry Bronsted what is an acid and a base?
Base accepts a proton, acid donates a proton when dissolved in water.
What is the part of the acid called after donating a proton called?
Conjugate base
What is the base called after accepting a proton called?
Conjugate acid
Strong acid and bases completely what?
Ionize in water
What are some strong acids?
Nitric, hydrochloric, sulfuric, perchlorate, hydrobromic, hydroiodic
What are poly protein/basic?
Acids and bases with two or more ionizable hydrogen/hydroxyl groups
What only ionizes partially?
Weak acids and bases
How do we define the strength of a weak acid?
The degree of dissociation or acid dissociation constant Ka.
Ka = ([H+][A-])/[HA]
Ka is a property of an acid or base at a given what?
Temperature
What is a buffer?
An aqueous solution that resists changes in pH upon the addition of an acid or base
What does a buffer consist of?
A pair of a weak acid and its conjugate base or a pair of weak base with its conjugate acid.
What is a substance that can act as an acid or a base?
Amphoteric
Significance of polyprotic substances?
Blood has a pH of 7.4 and is maintained by equilibrium between carbonic acid/bicarbonate/carbonate ions
Cells have a pH of 7.2 maintained by equilibrium between phosphoric acid in cells (dihydrogen/monohydrogen phosphate system
Is what amphoteric?
Yes
What is pKa?
When 50% of the molecule is dissociated
What is the optimum buffering capacity for a buffer?
About 1 pH unit from its pKa
What tend to be on the inside away from aqueous environments?
Hydrophobic amino acids like Phenylanine, Tyrosine, Tryptophan pKa ~11
What are some negatively charged residues of proteins?
Aspartame D and Glutamate D pKa ~5
What are some positively charge residues?
Lysine pKa~11 Arginine pKa~12.5 and Histidine pKa~6
You can estimate the charge of an amino acid if the pH is how many units away from the pKa?
2
What is the isoelectric point?
The pH at which all ionizable groups balance and the net charge is zero, for a protein
The analyses interacting the most what with the stationary phase will take longer to pass through the system?
Strongly
What is the strength of the analyte to interact with the stationary phase described by?
The parameter Kav or partition coefficient/distribution coefficient
Kav can be from what two values?
0 and 1
What is the distance distance moved by the substance divided by the distance moved by a solvent front called?
Retention time/retention factor
What are the classification of chromatography based on attractive forces?
Ion exchange anion/cation
Partition-non polar/polar
Size exclusion
Affinity
What are two types of resins?
Polystyrene and cellulose/dextran
What are the parameters of a chromatogram?
Retention time (depends on size of the column and flow rate)
In ion exchange chromatography (IEC), what is the stationary phase?
A matrix of beads functionalists with a charge
In IEC what is the mobile phase
A solvent with a salt/pH gradient buffer
In IEC useful buffers will be 1-1.5 units above or below the what of the protein
PI
Gel filtration separates based on what?
Size and shape of the compounds
For size affinity what is Kav?
Kav = (Ve-Vo)/(Vt-Vo)
If Kav is greater than 1 then the molecules is what?
Bound non-specifically to the column
If Kav is less than 0 then what?
There is a crack in the column
Ve/Vo is independent of column size and protein concentration but may be dependent on what?
Temperature for some proteins
Ve actually depends on the shape of the molecule there the dependence on MW is only what?
Approximate
What are advantages of gel filtration chromatography?
Doesn’t depend on pH, ionic strength and buffer composition.
What are limitation of gel filtration chromatography?
Sensitive to temperature and packing conditions
In affinity purification only molecules that have what will bind to the resin?
A complementary binding site
What are advantages of gene fusion or native
For tagged proteins
Easy purification >90% purity
Tagged protein can b used as a method of detection
Solubility and stability can be improved
Untagged proteins
Tag removal is not needed
Closer to native conditions
What are dissadvantages of tagged or native gene fusion?
For tagged proteins
Take my interfere with protein activity, structure, and function
Cleavage of rage may not reach 100% if cleavage site is buried
After cleavage the protein of interest may lose its solubility or remain permanently attached to the Tate due to protein-protein interactions
Untagged proteins
Purification and detection is not simple
What are applications of gel chromatography?
High resolution
MW estimation
