Tuesday, March 24, 2015

Determination of protein concentration

Introduction
Finding the exact quantity of proteins in a solution is very often necessary in the biochemical practice and to analyse clinical samples as well as in research. There are many ways to measure protein concentration. In chromogenic methods, the absorbance of a coloured product formed by the protein and an organic molecule is measured. Protein concentration can also be determined from the protein's own (intrinsic) UV absorbance. However, these methods may give different results for different proteins of the same concentration. Also, different methods can yield somewhat different results for the same protein. Also, dilution factor may be a important parameter.

Text source
There is no absolute photometric protein concentration assay. All methods have advantages and disadvantages and we must choose among them by taking the following aspects into consideration: specificity, sensitivity, the measurable range of concentration, the accuracy, the nature of the protein to be examined, the presence of materials interfering with the measurement, and the time required for the measurement.

Principle

Biuret test
Molecules with two or more peptide bonds react with Cu2+ ions in alkaline solution and form a purple complex. Nitrogen atoms of the peptide bonds form a coordination bond with the metal ion. The quantity of the complexes formed is proportional to the number of peptide bonds.

In practice, the determination of protein concentration is done using a calibration curve created using samples of known concentration. The protein treated with biuret reagent is measured at 540 nm after the purple product is formed.

The advantages of the method include that only few materials (e.g. Tris and amino acid buffers) interfere with it, it can be done in a short time and does not depend on the amino acid composition of the protein. Its disadvantages are its low sensitivity and that it requires at least 1 mg of protein.

Lowry (Folin) protein assay
This is sensitive technique where a coloured product is formed similarly to the biuret reaction, but a reagent strengthen the colour, which is kown as Folin–Ciocalteu reagent (a mixture of phosphotungstic acid and phosphomolybdic acid in the Folin–Ciocalteu reaction). The reaction mechanism is not well understood, but involves reduction of the Folin–Ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). The strong blue colour is created by two reactions: (1) formation of the coordination bond between peptide bond nitrogens and a copper ion and (2) reduction of the Folin-Ciocalteu reagent by tyrosine (phosphomolybdic and phosphotungstic acid of the reagent react with phenol). The measurement is carried out at 750 nm.

As in the biuret reaction, a calibration curve is created (for example using BSA, bovine serum albumin), and the concentration of the unknown protein is determined from the curve.

The advantages of the method include that it is quite sensitive and is able to detect even 1 µg of protein. Its disadvantages are that it takes rather long to carry out, is disturbed by various materials (including ammonium sulphate, glycine and mercaptans) and that the incubation time is critical. As different proteins contain different amounts of tyrosine, the amount of the coloured product will also be different. As a consequence, this method is more suited to compare the concentration of solutions of the same protein than to absolute measurement.
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Monday, March 23, 2015

List of Experiments for Biochemistry Laboratory work

                        List of Experiments for Laboratory work
Course Title: Laboratory work I: Biochemistry (Microbiology)
Credit: 1                                                                                                                                  

1. Preparation of normal, molar and percent solutions.
2. Preparation of different buffer solutions and measure their pH.
3. Determination of the isoelectric point of casein (of milk).
4. Titration of amino acids and find the physical constants.
5. Study the effects of temperature and pH on the enzymatic activity of salivary amylase.
6. Estimation of given protein concentration by Bradford, Lowry (Folin) protein assay and by Biuret test.
7. Qualitative detection of glucose in urine (the Trommer reaction).
8. Quantitative determination of glucose in blood by a glucose-oxidase method.
9. The determination of activity of the Krebs cycle dehydrogenases in the liver.
10. Determination of total cholesterol concentration in serum.
11. Determination of urea concentration in serum and urine.
12. Quantitative determination of phospholipids concentration in serum.
13. Determination of saponification value of the fat.
14. Separation of amino acids by chromatographic techniques and detection by nin-hydrin test. 

Tuesday, March 12, 2013

A Strategy Game in Linux Environment, 0 AD (zero-ey-Dee)

If you are fond of strategy game, 0 A.D. (zero-ey-dee) is one of the best game, which is available in Linux (also in Windows and Mac). It is a free, open-source, historical Real Time Strategy (RTS) game.

The game winning secrets resembles with the “age of empire 2,” i.e., build more and more modern weapons and attack as soon as possible. Defensive strategy hardly works.

However, currently it is available only in alpha version, by Wildfire Games. Even though it is in alpha phase. I have tested and it is working well in Ubuntu 12.04 LTS except the movement became very slow in some missions.

0 AD provides a platform not only for playing game, but also we can build a scenario and map ourselves. Here are some print-screens.







Monday, March 04, 2013

Is Debian the Most Stable Linux Distro?

I have been using Fedora for many years. Fedora is the leader out-breaking new software. I have found many research related software are more comfortable in fedora than some other Dostros, which I have tested. But the greatest drawback of this OS is the instability and crashing of several times. Surprisingly windows 7 showed great improvement, however, it is not freeware.

Because of YUM and RPM system and is bluish configured, I had shifted in CentOS. Although CentOS is designed for server, it was good in desktop also. And I could install research related software and they were working well, however, not good for Skype. But when I made update a few days after installing 6.3, the system did not start. There was no facility for bug report at that case. It booted only and hanged on log in screen. I had restarted several times but same problem persisted.

Then I tried Linux Mint. I surprised with its awesome feature. Really, it was very good looking. But yesterday when I updated Linux Mint 14, Nadia, I got the same problem as CentOS. Does it mean Linux OS are still in infancy in terms of reliability and stability.

Probably, I have to try Debian but may I install the latest version of some software such as Libreoffice, VLC, Autodock, Chimera, etc.

At this stage I need such Distro, which is stable for at least for 2-3 years without compromising for the latest software and security. I mean the software / application could be installable a few months later than the final release date, if not immediately. Please comment if you have any idea.

Wednesday, January 30, 2013

Online courses by reputed universities free of cost

No doubt, Nepal is one of the poorest countries, however, our education and courses, syllabus is still competitive. Further study in reputed university is very difficult for us because our government never supports for any scholarships, unlike other developing countries. Here are some websites where we can find some lectures offered by MIT, Harvard University, etc. These are absolutely free. Though, we have very slow internet and limited time of electricity supply, still we can try.
EdX (Most of the courses are about electronics, computer, physics, etc.)
Coursera (This offers a wide range of courses including physics, computer, biology, etc.)

Thursday, January 17, 2013

Lipid Structures

Some structures of lipid. Forget about exact geometric isomerism and stereocenter.

Simple Lipids

Prostaglandin

Tuesday, December 25, 2012

Scholarships in China for Nepalese

Three Gorges University (CTGU), Yichang, Hubei, China has offered the fellowships for Nepalese. This is  one of good options to pursue a degree in China. It will provide 10 scholarships each year including Hydro-power and electrical engineering (4), medicine (2), Chinese language (2) and Business Studies (2).  Prof. Dr. Krishna Adhikari and Ex-PM Madhav Kumar Nepal has facilitated for this MoU. How much stipend will they provide? Not confirmed yet. Source:Gorakhapatra

In addition, Chinese Academy of Science also provides PhD fellowships under TWAS-CAS scheme, which provides at least $500 per month and this is sufficient to cover a good living expenses. 

Saturday, November 10, 2012

How Does Bovine Serum Albumin Protein Look Like?

If you are a biologist, serum albumin protein is very common to us. It is one of the abundant proteins in our body as well as the component of egg. Here are the some pictures of the bovine serum albumin that I have drawn using a software and x-ray crystallographic data deposited in protein data bank (3V03).
Ribbon structure of bovine serum albumin in black background

Ribbon structure of bovine serum albumin in black background side view

Ribbon structure of bovine serum albumin (3V03) in white background front view

Ribbon structure of bovine serum albumin in white background side view

Ribbon structure of bovine serum albumin (3V03) in white background left view