Protein assay

Protein assay PowerPoint PPT Presentation

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Protein concentration determination. The perfect protein assay would exhibit the following characteristicsFastEasy to useSensitiveAccuratePreciseFree from interferences. Commonly used protein assay. Absorbance at 280 nmBiuret assayLowery methodCoomassie Brilliant dye binding assayBicinchoninic acid (BCA) method.

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Protein assay

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1. Protein assay Chao-Zong Liu, Ph.D. Department of Pharmacology and Institute of Pharmacology and Toxicology, College of Medicine, Tzu Chi University

2. Protein concentration determination The perfect protein assay would exhibit the following characteristics Fast Easy to use Sensitive Accurate Precise Free from interferences

3. Commonly used protein assay Absorbance at 280 nm Biuret assay Lowery method Coomassie Brilliant dye binding assay Bicinchoninic acid (BCA) method

4. Absorbance at 280 nm The simplest, most straightforward method In this assay, ultraviolet is absorbed by aromatic residues ( e.g. tyrosine) at a wavelength of 280 nm As a rule, OD=1 is about 1 mg/ml Non-destructive Range of sensitivity: 0.2-2 mg/ml

5. The drawbacks of absorbance 280 nm Residual protein solution left in the cuvette after measurement The buffers may have UV absorbing components Require expensive quartz cuvette and a photometer with UV wavelengths The UV absorbing characteristics of proteins vary widely 1 mg/ml Bovine serum albumin 0.7 Trypsin 1.6 Chymotrypsin 2.02

6. Biuret assay This assay is based on Cu2+ interaction with protein In alkaline solution, the copper ions form tetradentate complexes with opposite pairs of peptide bonded nitrogens These complexes produce a blue color that can be measured at 550 nm The reaction is dependent on in part on peptide bonds and not solely on amino acid moieties.

7. The drawbacks of Biuret assay Lacks sensitivity It requires a relatively large sample size Because large amounts of material are not always available, the Lowery assay, which uses the Folin reagent to increase sensitivity, was developed.

8. Lowery method This assay is essentially a biuret reaction that incorporates the use of of Folin-Ciocalteu reagent for enhanced color development Its ten times more sensitive than the biuret assay It is believed that the enhancement of the color reaction in the Lowery procedure occurs when the tetradentate copper complexes transfer electrons to the phospho-molybdic/phosphotungstic acid complex (Mo+6/W+6, Folin phenol reagent) Reduction of the Folin phenol reagent yields a blue color read at 750 nm Range of sensitivity: 5-100 mg/ml

9. Advantages: Reliable method for protein quantification Little variation among different proteins Disadvantages: Many interfering substances Detergents Carbohydrates Glycerol ….. Slow reaction rate (time required: 40 min) Instability of certain reagents Alkaline copper reagents is unstable and requires daily preparation The assay is photosensitive Proteins irreversibly denatured

10. Coomassie dye binding method This method is known as the Bradford method This assay is based on the immediate absorbance shift from 465 nm to 595 nm that occurs when Coomassie Brilliant Blue G-250 binds to proteins in an acidic solution The dye has been assumed to bind to protein via electrostatic attraction of the dye’s sulfonic acid groups. The Coomassie blue has been shown to interact chiefly with arginine residues, but weakly with histidine, lysine, tyrosine, tryptophan and phenylalanine residues.

11. Advantages Rapid (10 min) Sensitive ( 25-200 mg/ml) Disadvantages Some variability in response between different purified proteins Proteins used for this assay are irreversibly denatured

12. Bicinchoninic acid (BCA) method Proteins react with alkaline copper II to produce copper I. BCA then reacts with copper I to form an intense purple color at 562 nm The macromolecular structure of the protein, the number of peptide bonds and the presence of four amino acids (cysteine, cystine, tryptophan and tyrosine) have been reported to be responsible for color formation

13. Advantages Single reagent End product is stable Fewer interfering substances than Lowry assay Sensitive Standard assay: 10-1200 mg/ml Microassay: 0.5-10 mg/ml Disadvantages Slow reaction time (40 min) Proteins irreversibly denatured

14. Staining SDS-PAGE Separated Proteins with Coomassie Brilliant Blue and Silver Coomassie Brilliant blue staining < 0.1 mg 0.1g Coomassie Brilliant blue R-250 40 ml Methanol 7 ml Acetic acid 53 ml DD water

15. Silver staining 100 times more sensitive than Coomassie Brilliant blue Pre-fix Fix (glutaraldehyde) Wash (distilled water) Dithiothreitol (DTT) solution Silver nitrate solution Staining with developing solution (containing 3% sodium carbonate) Stop solution (2.3 M citric acid)

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