Lab 1 Flashcards
(11 cards)
What are proteins?
high molecular weight polymers of a.a
A.A linked by?
peptide bonds
2 types of proteins (think structure)
Fibrous: 2ndary stucture (via H bonding)
Globular: tertiary stucture (via R group interactions)
3 ways to measure protein [ ]? Explain
Biuret, Lowrey, UV spec
n the colorimetric tests (Biuret test, and Lowry test which is slightly more sensitive so lower protein concentrations can be detected), copper ions form a purple-coloured complex with peptide bonds in an alkaline solution. This coloured complex can then be detected by measuring the absorbance in a spectrometer at either 540nm for the Biuret test or 500nm for the Lowry test . Free amino acids don’t react and since this test is the same for all proteins, a known protein can be used as a standard to create a concentration curve so determine the concentration of the unknown protein. For the Ultraviolet spectroscopy test , the amino acids tryptophan, tyrosine, and (lesser) phenylalanine which absorb light in the UV spectrum and are common in most proteins, are used to measure light absorbance at 280nm. Using a concentration curve, the concentration of the unknown protein can be found.
a.a in proteins that absorb light in UV spec
tryptophan, tyrosine, phenylalanine
what is the normal standard protein used as standards?
Albumin
Briefly explain the principle behind the colorimetric tests you used
In the colorimetric tests (Biuret test, and Lowry test which is slightly more sensitive so lower protein concentrations can be detected), copper ions form a purple-coloured complex with peptide bonds in an alkaline solution. This coloured complex can then be detected by measuring the absorbance in a spectrometer at either 540nm for the Biuret test or 500nm for the Lowry test . Free amino acids don’t react and since this test is the same for all proteins, a known protein can be used as a standard to create a concentration curve so determine the concentration of the unknown protein.
explain the principle behind the UV method you used.
For the Ultraviolet spectroscopy test , the amino acids tryptophan, tyrosine, and (lesser) phenylalanine which absorb light in the UV spectrum and are common in most proteins, are used to measure light absorbance at 280nm. Using a concentration curve, the concentration of the unknown protein can be found.
how you would measure protein in the following samples:
i) a small volume of sample that you are trying to keep for a future experiment
and which has a protein concentration of around 0.05 mg/ml.
ii) the protein in solution is known to be deficient in tyrosine.
iii) lots of sample, and an approximate protein concentration of 10 mg/ml.
iv) a sample which is turbid (free amino acids present), with a protein concentration
of around 1 mg/ml.
i) the uv test, cause low [] and the sample has to be revsed points to the uv test, no reagents are added to the smaple.
ii) Either of the colorimetric tests would work for this (Biuret or Lowry). UV would not work because it needs a tyrosine to absorb light in the UV spectrum range. With a deficient of tyrosine, the test would not work properly.
iii) All three of the tests (Biuret, Lowry and UV spectroscopy) would work perfectly fine for lots of sample and a 10mg/mL concentration.
iv) Either of the colorimetric tests (Biuret or Lowry) because free amino acids don’t react in these tests. Only the peptide bonds.
(a) What was the purpose of making three dilutions (undiluted, 2-fold, and 10-
fold) of the unknown protein solution?
The purpose of making the three dilutions for the unknown is that we’re unsure of what the concentration of the solution is. It could very well fall outside of the dilute standards we’ve graphed so by making dilutions, we increase the chance that at least one of our unknown dilutions will be on the graph.
If you need a final volume of 3 mls of the unknown solution in order to take an absorbance reading in a spectrophotometer, how would you prepare the unknown dilutions? In other words, how much water and how much of the unknown solution would you need to pipette into a test tube to end up with the proper volume (3 mls) and the proper dilution (2-fold and 10-fold)? HINT: Keep in mind that a 2-fold dilution is equal volumes of distilled water and the unknown solution and a 10-fold dilution is 1 volume of the unknown solution to 9 volumes of distilled water.
For the 2-fold dilution, in order to make 3mLs, I would add 1.5ml of water and 1.5ml of the unknown protein solution. For the 10-fold dilution, in order to make 3mLs, I would add 2.7mLs of water and 0.3 mLs of unknown protein solution
