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Detection and quantification of proteins

Detection and quantification of proteins. MALIK ALQUB MD. PHD. Performance of a test . Sensitivity of a test. Ability of a test to identify correctly affected individuals proportion of people testing positive among affected individuals . True patients (gold standard). + －.

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Detection and quantification of proteins

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  1. Detection and quantification of proteins MALIK ALQUB MD. PHD.

  2. Performance of a test

  3. Sensitivity of a test • Ability of a test to identify correctly affected individuals • proportion of people testing positive among affected individuals True patients (gold standard) + － True positive (TP) Test False negative (FN) Sensitivity (Se) = TP / ( TP + FN )

  4. Sensitivity of a PCR for congenital toxoplasmosis Sensitivity = 54 / 58 = 0.931= 93.1 %

  5. Specificity of a test • Ability of test to identify correctly non-affected individuals • - proportion of people testing negative among non-affected individuals Non-affected people + － False positive (FP) Test True negative (TN) Specificity (Sp) = TN / ( TN + FP )

  6. Specificity of a PCR for congenital toxoplasmosis Specificity= 114 / 125 =0.912 = 91.2 %

  7. Performance of a test Disease Yes No + TP FP Test ­ FN TN TP Se = TP + FN TN Sp = TN + FP

  8. Distribution of quantitative test results among affected and non-affected people(ideal case) Non affected: Threshold for positive result Affected: Number of people tested TN TP 0 5 10 15 20 Quantitative result of the test

  9. Specific assay Specificity A specific assay gives a response to the substance being assayed and to no other substance. Any other substance producing the same response as the substance being assayed is called an "interfering substance". Very few assays are 100% specific; virtually all respond to some interfering substance.

  10. Specific assay Benedict's reagent Benedict's reagent is used as a test for the presence of reducing sugars such as glucose, fructose, galactose, lactose and reducing disaccharides Then the test is specific for reducing sugars. But if we want to use it to detect monosaccharides then it would react with disaccharides. We can say the test is not specific for monosaccharide.

  11. Detection of proteins

  12. Proteins: Common properties • In spite of functional differences between the various proteins, they have certain common biophysical and biochemical properties. These include: • a basic composition of carbon, hydrogen, nitrogen and oxygen; • a backbone of covalent peptide bonds which join the amino acid units together; and • absorption maxima in the ultraviolet region.

  13. The Biuret Method • The Biuret reagent is made of (NaOH) and copper (II) sulfate (CuSO4), together with potassium sodium tartrate (KNaC4H4O6). • A blue reagent which turns violet in the presence of proteins, • The Sodium hydroxide does not participate in the reaction at all, but is merely there to provide an alkaline medium so that the reaction can take place.

  14. Principle: Biuret Method • Peptide bonds of proteins react with tartrate-complexed cupric ions in alkaline solutions to form a colored product. • In a positive test, a copper(II) ion is reduced to copper(I), which forms a complex with the nitrogens and carbons of the peptide bonds in an alkaline solution. • A violet color indicates the presence of proteins. • It is possible to use the Biuret reaction to determine the concentration of proteins because (for most proteins) peptide bonds occur with approximately the same frequency per gram of material.

  15. The intensity of colaration depends on protein amout High protein concentration the intesnsity of violet color would increase

  16. Biuret Method • The intensity of the color varies according to protein • concentration, and hence the absorption at 540 nm, is directly • proportional to the protein concentration, and can be determined • spectrophotometricallyat 540 nm. • Using the Biuret Assay to determine protein concentration in an unknown sample requires, as a preliminary, construction of a standard curve made by assaying a series of protein solutions with known concentration. The protein bovine serum albumin (BSA) is frequently used for constructing standard curves because of its availability and low price.

  17. Quantification of proteins Standard amount of protein. We know exactly how much protein in each well. Unknown concentration 1g 2g 3g 4g 5g

  18. the intensity of color is proportionally dependent with absorbed light; Or optic density Detector

  19. Standard curve • We draw the standard curve, concentration vs. Optic Density. • Then we measure the optic density of « unknown » • If optic density is known of the standard curve then we can find the concentration of the unknown optic density 1g 2g 3g 4g 5g concentration

  20. BioRad DC Protein Assay

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