UV Absorption Method of Protein Estimation | Protein Assay | Dr. Nagendra Singh | PENS#37
HTML-код
- Опубликовано: 7 авг 2024
- UV Absorption method of Protein Estimation | Protein Assay | Dr. Nagendra Singh | PENS#37
#proteinassay #UVAbsorption #proteinassay
UV absorption method is the simplest and most direct assay method for protein concentration determination in solution is to measure the absorbance at 280 nm in UV range. Amino acids containing aromatic side chains mainly Tyrosine and Tryptophan exhibit strong UV-light absorption. Proteins and peptides absorb UV-light in proportion to their aromatic amino acid content and total concentration.
The main methods of protein estimation are:
1. UV based detection
2. Biuret method
3. Lowry method
4. BCA method
5. Bradford method
The Lecture is given Dr. Nagendra Singh aiming to spread basic understanding and awareness of biological molecules and reactions. Dr. Nagendra Singh finished his Doctoral and Post Doctoral studies from "All India Institute of Medical Sciences" (AIIMS) New Delhi. Dr. Singh has a vast experience of research and Teaching in the fields of Structure & Molecular Biology and Biochemistry, which also include Biomolecules, Proteomics, Protein Engineering and Drug Design.
Dr. Nagendra Singh currently works at the School of Biotechnology, Gautam Buddha University, Greater Noida, INDIA (www.gbu.ac.in/FacultyProfiles/.... He does research in Molecular Biology, Structural Biology and Biotechnology. His current research interest include 'Structural and functional characterization of proteins involved in RNA metabolism from human and microbial origin. Dr Singh has published over 70 research articles in intentional Journals of good repute. He has over 20 awards and honors on his name. Few of the awards include "National Academy of Sciences of India (NASI, Allahabad) young scientist award", UGC-NET, CSIR-JRF, GATE, Best Research Awards etc. Dr. Singh has participated and presented research work at over 50 national and International conferences world wide.
Related Videos:
Top Five Protein Estimation Methods | Preparation of Standard Curve | Dr. Nagendra Singh | PENS#36
• Top Five Protein Estim...
UV Absorption Method of Protein Estimation | Protein Assay | Dr. Nagendra Singh | PENS#37
• UV Absorption Method o...
Biuret Test for Proteins | Protein assay | Estimation of Protein | Dr. Nagendra Singh | PENS#38
• Biuret Test for Protei...
Lowry Assay for Protein Estimation | Folin Lowry Method | Dr. Nagendra Singh | PENS#39
• Lowry Assay for Protei...
BCA Method of Protein Estimation | Protein Assay | Dr. Nagendra Singh | PENS#41
• BCA Method of Protein ...
Bradford Protein Assay | Protein Estimation | Dr. Nagendra Singh | PENS#42
• Bradford Protein Assay...
Thankyou sir
Nice
Why is the width of the cuvette needed for the calculation of the protein sample?
because how long radiation travels in solution would affect absorbance as per beers lamberts law
Does pka of protein affects UV absorbtion?
cysteine which consist of sulphur in its side chain, absorb to a lesser extent at 280nm. so can the methionine which also consist sulphur, absorb at 280 nm?
yes, met also absorbs but lesser than disulphide bond
@@proteinengineering007 okay sir
Does the concentration of a sample affect the absorbance measurement in the visible region?
@Upasana Shukla beers lamberts law is applicable to all range of radiations
Why only monochromatic light is used in beer Lambert law?
because a chromophore absorbs maximum at lambda max only..
Why phenylalanine (being an aromatic amino acid) absorbs least amount of UV rays & is not involved in the calculation of molar extinction coefficient.???
In the near-UV the molar absorbance
of phenylalanine is much smaller than that of tyrosine and tryptophan, and the spectrum of a protein between 240 and 300 nm is therefore dominated by the Tyrosine and Tryptophan sidechains.
Phenylalanine residues contribute fine structure to the spectrum between 250 and 260 nm.
What are the limitations to UV absorption when estimating protein concentration?
Since a number of aromatic amino acids vary from one protein to another so thats why it gives very high variability, so we can say its not a very accurate method.
Also the presence of detergents and denaturing agents affects its accuracy as they can be absorbed at the same wavelength.
@@ibt007gaurisingh4 true
Sir ph absorbance ko affect karega?
jaise jaise solution ki ph values badegi,uski vjha se solution me protonated ions zyada honge.Jiski vjha se maximum absorbance hoga jb vo light absorb krega
@@aaradhyadev6692 yes..ph affects absorption. the change in absorption would depend on nature of chromophore. for example absorption spectra of deprotonated shifted to higher wavelenths.
How do protein absord uv light?
Due to the presence of tyrosine and tryptophan, proteins and peptides containing these aromatic amino acids absorb UV light at a wavelength of 280 nm.
Mention the causes why Beer Lambert law is not applicable at higher concentrations?
Beer Lambert Law is not applicable for higher conc. because the results may deviate due to interactions between the absorbing species (because of close proximity) which add to alterations of the refractive index of the medium.
Does pH affect absorbance?
As solutions rise in pH values, there are more protonated ions in the solutions, thus raising the maximum absorbance as they absorb light.. The pH of 5.397 is higher on the side of lower wavelengths and decreases in values for molar extinction coefficient for the range of higher wavelengths.
This method is based on which law?
beer lamberts law
sir sample ki concentration affect kregi absorbance ko?
Beer lamberts law follow hoga jese jese conc. Badhegi vese vese absorbance badega
@@namanarora1507 true
Why do proteins absorb at 280 nm
Absorbance of proteins at 280nm is primarily caused by presence of the aromatic amino acids tryptophan and tyrosine...
Peptide bonds are primarily responsible for the peak at 200 nm
Which amino acids can absorb UV and why?
All aromatic amino acids absorb ultraviolet light