DIFFERENT METHODS OF PROTEIN ESTIMATION - PROTEINS AND ENZYMES ASSIGNMENT

A brief PROTEINS AND ENZYMES ASSIGNMENT on the topic - " DIFFERENT METHODS OF PROTEIN ESTIMATION " . Includes Methods, Applications, Uses and different techniques of protein estimation and separation . Separation on the basis of charge

1. “ DIFFERENT METHODS OF PROTEIN ESTIMATION “ PROTEINS AND ENZYMES ASSIGNMENT MADE BY :- “ RISHABH SHARMA ”

2. BIciNCHONINIC ACID :- 1) This colorimetric, two-step assay was originally developed in 1985 – Like the Lowry assay, the first step here is to complex the protein with copper ions. In the second step, this protein-bound copper chelates BCA to give an intense purple color. Conveniently, the purple’s intensity is linear with the amount of protein. Less conveniently, each sample’s intensity must be compared to a standard curve because (unlike the Folin-Lowry method) this assay doesn’t have a set endpoint.

3. BICINCHONINIC ACID : CHEMISTRY :- While slower than the Bradford, the BCA assay is a great option if your protein samples contain > 5% detergents.

4. 2) BIURET METHOD :- Sensitivity of this method is very low. It requires high protein levels from 1-20 mg of protein. The reagent used in this method are Copper sulfate, Sodium hydroxide, and Potassium Sodium Tartrate. Protein reacts with this alkaline copper complex and color changes to violet. The protein can then be estimated by reading the absorption at 540nm. This method takes 20-30 minutes to complete. This method does not rely on amino acid composition and hence can measure all protein samples with accuracy. The main disadvantage of this method is that buffers with Tris, Ammonia interferes with the reaction.

5. BIURET METHOD :-

6. BIURET METHOD : CHEMISTRY :-

7. BIURET METHOD : FORMATION OF Cu COMPLEX :-

8. 3) UV ABSORPTION :- Sensitivity of this method is moderate. It can detect proteins in the range of 50-100 µg. In this method, no reagents are required, the liquid protein sample is monitored under UV absorption at OD 280nm. Recently, simple machinery like Nanodrop and pico drop are used where even 1 µl of the protein sample is enough to determine protein concentration. This method takes less time, within 10 minutes.

9. 4) LOWRY ASSAY :- Lowry assay is one of the old methods used for protein estimation developed by Oliver Lowry (1951). This is a highly sensitive and gives accurate results. It detects proteins at low concentrations of 2-5 µg. In this method, first, the copper ions are reduced under alkaline conditions and forms a complex with peptide bonds of the protein. This complex then reduces Folin-Ciocalteau reagent and results in the change in color to deep blue and absorption can be measured at 650-750nm. It takes about 45-60 minutes for assay completion.

10. LOWRY METHOD : - “ A BRIEF OVERVIEW “

11. LOWRY ASSAY MECHANISM OF ACTION :-

12. 5) UV ABSORPTION :- Simple but often unreliable, this method estimates the amount of protein by measuring the characteristic absorption of tyrosine and tryptophan at 280 nm. The advantages of this method are that it is simple, and the sample is recoverable. The method has some disadvantages, including interference from other chromophores, and the specific absorption value for a given protein must be determined. “The extinction of nucleic acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and hence, a few percent of nucleic acid can greatly influence the absorption.”

13. 5) UV METHOD ABSORBANCE GRAPH

14. 6) BRADFORD ASSAY :- This method was developed by Marion M Bradford (1976) and widely used. This is a very sensitive method and simple dye binding assay. This method uses Coomassie brilliant blue-250 dye that binds with negatively charged protein molecules. The dye color changes based on protein concentrations and the absorption is measured at 595nm. This method takes very less time compared to other regent based assays. Within 10-15 min the results can be obtained. Using standard curve protein concentration can be estimated in no time.

15. 6) BRADFORD ASSAY :- PRINCIPLE

16. PROTEIN FRACTIONATION :- SEPARATION USING CHARGE

17. REFERENCES :- https://www.analiticaweb.com.br/newsletter/16/51859_proteina_biureto.pdf https://www.researchgate.net/publication/230127798_Salt_fractionation_of_plasma_proteins_A_procedure_to_teach_principles_of_protein_chemistry https://www.ncbi.nlm.nih.gov/pubmed/388794 https://www.oxfordreference.com/view/10.1093/oi/authority.20110803100438675 https://www.chegg.com/homework-help/questions-and-answers/principle-behind-salt-fractionation-proteins-q15094071 https://www.sciencedirect.com/science/article/abs/pii/S0003269708000389

18. THANK YOU :)