proteins Flashcards
(18 cards)
why is the determination of protein concentration in a sample important in forensic science
it can be used as a diagnostic tool
what is the simplest way to estimate protein concentration and what is its problem
to measure absorbance at 280nm due to the aromatic amino acids - however, there are many other compounds that absorb in this region, so it can only be used on pure protein samples
name 3 methods of determining protein concentration
- Folin-Ciocalteau (Lowry)
- Bradford
- Biuret
explain the chemical basis of the Folin-Ciocalteau (Lowry) method
the phenolic groups in tyrosine and tryptophan residues form a blue-purple complex with the Folin reagent - these complexes absorb at 660nm
explain the chemical basis of the Bradford method
Coomassie Brilliant blue G250 dye binds to proteins and then absorbs at 595nm
explain the chemical basis of the Biuret method
under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. absorbance measured at 540nm
whichever method used to determine the concentration of the protein in a sample what else must be produced
a standard curve using a solution of protein (often bovine serum albumin) of a known concentration
which of the three methods did we use in the practical
Biuret
what type of samples did we determine the protein concentration of
plasma
what is the normal value of plasma proteins in humans
6-8 g/100ml (60-80 mg/ml)
in what cases does a plasma sample have a lower than normal concentration
malnutrition, liver disease or severe burns
in what cases does human plasma have a higher concentration of protein to normal
dehydration due to severe vomiting or diarrhoea
what was the concentration of our protein standard
10mg/ml
why do we 1 in 10 dilute the samples
as our standard curve covers a 1-10mg/ml range and the samples are 60-80mg/ml so we need to dilute them to be 6-8mg/ml
what piece of equipment is used to determine the absorbance and at what setting is it at
spectrophotometer at 540nm
why do we use the same cuvette for all standard solution tubes but not sample tubes
using the same cuvette for standard solutions is to minimise cuvette variance, but with samples we do not want to change the concentrations from solution being left in the cuvette sop we change it. also prevents cross-contamination
what do we plot after reading all absorbances
the standard curve of absorbance against protein concentration
once calculated the protein concentration what do you have to do to conclude the actual concentration in the sample
multiply by 10 due to the earlier dilution
