Protein practical Flashcards
(20 cards)
The simplest way of estimating protein concentration
Measure the absorbance at 280 nm due to the aromatic amino acids (primarily tyrosine and tryptophan)
However , other compounds absorb in this region, so this is only useful when dealing with pure protein samples
Folin-Ciocalteau (Lowry) method for estimating protein concentrations
The phenolic groups of tyrosine and tryptophan residues from a blue-purple complex with the Folin reagent. These complexes absorb at 660nm
Bradford method for estimating protein concentrations
Coomassie Brilliant Blue G250 dye binds to proteins and then absorbs at 595nm
Biuret method for estimating protein concentrations
Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. Absorbance measured at 540nm
Determining the concentration o protein in a sample
Whichever method is used, it is necessary to prepare a standard curve using a solution of protein (often bovine serum albumin) of a known concentration. This can then be used to determine the concentration of protein in a sample
The normal value of plasma proteins in humans:
6-8 g/100ml
OR
60-80mg/ml
hypoproteinaemia
In some cases the value of plasma proteins is lower than normal; this is known as hypoproteinaemia and can be caused by malnutrition, liver disease or severe burns
Hyperproteinaemia
In some cases, the value of plasma proteins is higher then normal; this is known as hyperproteinaemia and can be caused by dehydration due to severe vomiting or diarrhoea
Materials
> Biuret reagent
Protein standard ( bovine serum albumin (BSA); 10mg/ml)
Distilled water
Protein samples of two suspected victims
Health and safety
Wear safety glasses and dispose of biuret solutions in the bottle in the fume hood.
Be careful with your pipetting.
Pipette protein solutions slowly/gently
Step one
To prepare the standard curve, mix protein standards and water. Calculate the protein concentration values use C1 x V1= C2 X V2
Protein (BSA) standard (ml) should increase as water decreases (ml)
Step two
Dilute each of the 2 plasma samples 1 in 10 with distilled water. You will need 3 ml
of each sample. Be sure to mix each diluted sample by inverting the tube a few
times or pipetting the solution up and down gently.
Step three
You are going to measure the absorbance of each diluted sample in duplicate.
Therefore, pipette 1 ml of diluted sample A into a tube, and 1 ml into another tube.
These are ‘twin daughters’. You can label the tubes e.g. A1, A2. Discard the residual
dilution left over in the original ‘mother’ tube. Repeat for samples B, labelling the new
tubes B1 and B2.
Step four
Discard the original ‘mother’ tubes.
Step five
You should now have 8 standard tubes and 4 plasma sample tubes all containing 1
ml of solution (roughly the same height).
Step six
Add 3 ml of Biuret reagent to each tube and allow to stand for 20 min (at RT)
Step seven
Transfer app. 1ml from the standard tube 1 into a cuvette and use this to zero
(blank) the spectrophotometer.
Step eight
Return the measured standard solution 1 back to tube 1, and using the same cuvette (to minimise the effect of cuvette variance), read the absorbance of each of the standards in
tubes 2-8, working low to high protein concentration.
Step nine
Using a fresh cuvette in each case (zeroed with tube 1 as above), read the
absorbance of each of the two samples in tubes A1, A2, B1, and B2. So you have
two replicates of the each sample so 2 x 2 = 4 tubes in total to measure
Analysis of results
- Plot the standard curve of absorbance against protein concentration. Try to utilize the whole A4 as this gives the best accuracy
- Calculate the concentration of protein in each of the plasma samples.
- What can you conclude regarding the health of the two suspected victims?
- Are all two results equally accurate? If not, why not?
