Nuclear Magnetic Resonance (NMR)

NMR is a technique vastly used for studying biological molecules, crystalline structures,
molecular physics and non-crystalline structures. It is based on the fact that all nuclei that have odd number of protons have a magnetic field  and electromagnetic pulse or pulses. Nuclei absorb energy from these electromagnetic pulses and then rediate it back in the form of resonance frequency. The strength of resonance frequency is directly proportional to the strength of applied electromagnetic pulses. So, this allows scientists to exploit this property of magnetic nuclei to suty quantum mechanical magnetic properties of an atomic nucleus. NMR spectrometry is also used in advanced medical imaging techniques such as magnetic resonance imaging (MRI).

The principle of NMR usually involves two sequential steps
The alignment (polarization) of the magnetic nuclear spins in an applied, constant  magnetic

The perturbation of this alignment of the nuclear spins by employing an electro-magnetic,
usually radio frequency (RF) pulse. The required perturbing frequency is dependent upon
the static magnetic field (H0) and the nuclei of observation.

Both the fields are set perpendicular to each other to give a better NMR signal. Both
use intense magnetic fields to deliver better quality spectra result of which is
detailed in zeeman effect, chemical shifts and knight shifts (in metals).

Analytical Utracentrifuge
Analytical Ulracentrifuge is widely used in studying molecular biology, proteins and long
polymers since long. A sample is taken in an Ultracentrifuge and accelerated to a very high speed of about 3600 kms2. The sample is spun with such a high acceleration and optical detectors are used to examine the sample using ultraviolet absorption or interference optical refractive index sensitive system. The sample is monitored in real time by using the above two methods. Two types of experiments are usually performed on these instruments

Sedimentaion velocity Experiments

Sedimentaion equilibrium Experiments
Sedimentation velocity experiments are concerned the concentration distribution to find the molar mass and size of the dissolved biological macromolecules. The size resolution for this method is the square of the radii of the particle being examined,or it can be adjusted by rotor speed of experiment.

Sedimentation equilibrium Experiments are performed without having regard to concentration
ditribution of the particle dissolved but are, rather concerned with the final  steady-state of
experiment, where sedimentation is balanced by diffusing opposed to concentration gradient in
sedimentation velocity experiments.

Basic Electron Microsopy
Electron microscopy is the most powerful device for examining cellular objects, biological
molecules and other small objects that are not easily imageable using optical microscopy. In this
case, a high beam of charged electron is projected on a specimen to illuminate and find its image to a very high resolution ever possible by 1,000,000X whereas optical microsopy is only able to 1,000X. It is because of the fact that electrons ar too small as compared to photons that are used in optical microscopy.
Cyro-Electrom microscopy

Cyro-Electrom microscopy is a type of electron microscopy but in cyrogenic
temperatures,usually in liquid nitrogen temperatures. Cyro-Electrom microscopy is helpful in
studying proteins and other biochemical processes in its actual physiological environment unlike X-rat crystallography which have a retrospective impact on the sample studied because of the
articficially created environment.. Thats why cyro-Electrom microscopy is more valuable than
other technigues.

Negative Staining
Negative staining is an established method especially in diagnostic microscopy where there
is a thin specimen to be contradted with an optically opaque fluid. A negative staining technique
uses an ink to be sprinked over the sample like nigrosin and then to dry the sample,after which it is undergone an electromicroscopic treatment. The target material in the sample is seen light in contrast to the dark background.

Single Particle Analysis
Single particle analysis and electron tomography are the tools used in the structural biological analysis of proteins and other organic macromolecules. Single particle analysis in
transmission electron microscopy examines macromolecules and large proteins as opposed to
electron tomography which observes the sub-cellular portions of the organism. Electron
tomography when used as a single particle anaysis will bridge the gap between optical analysis andelectron microscopic analysis of macromelecules and other sub-cellular organelles.

Mass Spectrometry
Mass spectrometry is an analytical techniques used in the determination of biological
molecules structures and the elemental composition of organic biological moecules such as protein  etc. It employs the method of ionizing the sample and then passing it through an electric field. The charged ionized particles are deflected in the electric field based on their mass to charge ratio.

Newton second law of motion is applied in the deflection movement phenomenon.

Circular Dichroism
CD spectroscopy is relatively new technique which is based on the difference in the
absorption of left and right circularly polarized light. The phenomenon is seen in the absorption
bonds of optically chiral molecules.It has wide range of applications including structural analysis
of biological molecules etc.

Sodium dodecyle sulphate polyacrylamide gel electrophoresis is a technique widely used in
biological forensics,protein isolation based on protein electrophoretic mobility. Electrophoretic
mobility is a function of length of polypeptide or weight of polyprotein.

In a nutshell, there are growing numbers of techniques in use today by reseachers and
biologists to delve deep into the protein structure and function to get fully acquainted with the life
most important constituent, the Protein. Research is currently under process to know more about the protolytic enzyme that cause Hapatitus C Virus using multiple methodologies. Although we were not able to mention the full set of techniques used in proteinology, but we did make an excellent effort to describe the overall structural based and non structural based techniques to a great deal. Yet  more and more techniques are being discovered to cover the subject of protein studies in great detail. We wish good luck to all the researchers in this field.


Post a Comment