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25 Cards in this Set
- Front
- Back
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What type of analytical techniques is the Bradford Reagent? What is it used to measure? |
Spectroscopic Measure concentration of protein in solution |
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The colourimetric assay is based on the absorbance shift of what dye? Maximum absorbance of this dye occurs at what wavelength? The absorbance of this dye depends on what? |
Coomassie Brilliant Blue 595 nm Amino acid composition of protein |
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The shift from red dye, to Coomassie Brilliant Blue, involves 3 stages, with the transition from stage 2 to 3 involving proteins. State the 3 stages involved. |
Cation doubly protonated Neutral singly protonated Anion unprotonated |
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The dye, Coomassive Brilliant Blue reacts mainly with arginine. What 5 amino acids does it react less well with? What 2 types of proteins is it less accurate for? |
Histidine, lysine, tyrosine, tryptophan and phenylalanine Acidic and basic (need neutral) |
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Monoamine oxidases catalyse what? Where are they found? |
Oxidation of monoamines Outer membrane of mitochondria |
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State 3 places where MAO-A is found. Where is MAO-B found? |
MAO-A: Liver, GI and placenta MAO-B: Placenta |
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MAO is required for the inactivation of monoaminergic neurotransmitters. State 3 types of these. |
Seratonin, melanonin and adrenaline |
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Complete this equation: RCH2NH2 + H2O + O2 ---(MOA)--->? + ? + ? What does a change in absorbance signify? |
RCHO + NH3 + H2O2 Amine used, aldehyde produced |
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In what reaction pathway does PGK have a use? Where is it found? A deficiency is often X linked recessive. What 3 disorders are produced as a result? What can over expression of PGK cause? |
Glycolysis Cytosol Haemolytic anemia, mental disorder and myopathy Gastric cancer |
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Complete the following stages in the PGK reaction: ?<--(PGK)(ATP->ADP)-->?<--(GADPH)(NADH+H+->NAD++Pi)-->? |
3-Phosphoglycerate 1,3-bisphospho glycerate Glyceraldehyde 3-Phosphate |
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NADH absorbs strongly at what wavelength? NAD+ doesn't absorb strongly here |
340 nm |
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Protein absorb strongly at what wavelength? What is this due to? |
280 nm Pi conjugated systems of phenylalanine, tyrosine and tryptophan |
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What wavelength do nuclei acids absorb strongly at? Why is this? |
260 nm Adenine, guanine, cytosine and thymine |
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State 3 things that absorb strongly at 230nm? |
Peptide bonds, phenol and sugars |
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In the r^2 equation from the Bradford Standard Curve, the units of X are? How are these converted into microg/microL? |
Microg/milliL Divide X by 1000, to get (mg/mL), then times by 250 undo dilution factor |
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In the experiment, what was the difference between the Odd and Even tubes? How was activity calculated? |
Even tubes: no substrate in them Odd-Even=activity (Delta A) |
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What do the following symbols represent? I0, I, A, c, l, n and epsilon |
I0: Incident intensity I: Transmitted intensity A: Absorbance c: Concentration (molL^-1) l: Length of solution the light has passed through (cm) n: Number of moles (M) Epsilon: Molar absorptivity constant |
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State the Beer-Lambert law as an equation. How can this be rearranged to obtain the expression for molar absorptivity? |
A=log10(I0/I)=epsilon.l.c Epsilon: A/lc |
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State the equation usedd to calculate the concentration od aldehyde. |
Caldehyde= DeltaA/epsilon.l |
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Activity is measured as the number of moles/time. How is naldehyde calculated? What must be done to get the specific activity of the protein? How is mass calculated? What are the units for specific activity? |
naldehyde= C.V Divide by the amount of protein you added (mg) Mass=C.V micromol/min/mg |
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In order to calculate the amount of NADH used, the equation A=epsilon.l.c can be rearranged to produce? What does A represent? What are the units of C...? What are the units of naldehyde/nNADH? |
CNADH(used)=A/epsilon.l A: Activity of enzyme M/min micromol (so times by 10^6) |
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There are 7 potential sources of error in the experiment. State 5 of these. |
Answer can include any 5 of the following: Standard curve isn't linear Extrapolation errors Timing Pipetting Miss labelling Bradford reagent Cell fractionalisation |
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The homogenate is comprised of the nuclear fraction, the mitochondrial fraction and the cytosolic fraction. What percentage of protein (homogenate being 100%) is found in each fraction? |
C= 30% + 50% + 80% |
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State the units used to measure the following measurments: Delta A Caldehyde etc naldehyde etc Activity Mass of protein Specific activity |
No Units M micromol micromol/min mg micromol/min/mg |
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What should you do if the Even recording is>than the Odd recording? Which fraction should have the highest activity? |
Record as zero Fraction M |
