1.Protein Estimation
Protein determination is necessary to estimate the amount of protein
in the sample, to normalise against the protein concentration
or during purification procedures. Depending on the amount of
sample, accuracy and presence of interfering agents, one needs
to decide on the method to be used. For accurate quantification,
the sample protein is compared with a known amount of a standard
protein which could either be the commonly used bovine
serum albumin (BSA) or it could sometimes be immunoglobulin
G (IgG). The various methods and their specifications are outlined
below:
1.1 Absorbance Assays
The aromatic rings in the protein absorb ultraviolet light at an
absorbance maximum of 280 nm, whereas the peptide bonds
absorb at around 205 nm. The unique absorbance property of
proteins could be used to estimate the level of proteins. These
methods are fairly accuratewith the ranges from 20μg to 3mg for
absorbance at 280 nm, as compared with 1 to 100μg for 205 nm.
The assay is non-destructive as the protein in most cases is not
consumed and can be recovered. Secondary, tertiary and quaternary
structures all affect absorbance; therefore, factors such as
pH, ionic strength, etc can alter the absorbance spectrum. This
assay depends on the presence of amino acids which absorb UV
light (mainly tryptophan, but to a lesser extent also tyrosine).
Small peptides that do not contain such amino acids cannot be
measured easily by UV.
Requirements
- Quartz Cuvettes
- UV-Spectrophotometer
Free Radicals Defined:
Free radicals are a byproduct of normal cell function. When cells create energy, they also produce unstable oxygen molecules. These molecules, called free radicals, have a free electron. This electron makes the molecule highly unstable. The free radical bonds to other molecules in the body – causing proteins and other essential molecules to not function as they should. Luckily, antioxidants can minimize free radical damage.
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Although scientists as far back in history as Aristotle recognized that the features of one generation are passed on to the next (…like begets like…) it was not until the 1860′s that the fundamental principles of genetic inheritance were described by Gregor Mendel. Mendel’s work with common garden peas, pisum sativum, led him to hypothesize that phenotypic traits (physical characteristics) are the result of the interaction of discrete particles, which we now call genes, and that both parents provide particles which make up the characteristics of the offspring. His theories were, however, widely disregarded by scientists of the time. In the last quarter of the 19th century, however, microscopists and cytologists, interested in the process of cell division, developed both the equipment and the methods needed to visualize chromosomes and their division in the processes of mitosis (A. Schneider, 1873) and of meiosis (E. Beneden, 1883).
As the 20th century began many scientists noticed similarities in the theoretical behavior of Mendel’s particles, and the visible behavior of the newly discovered chromosomes. It wasn’t long before most scientists were convinced that the hereditary material responsible for giving living things their characteristic traits, and chromosomes must be one in the same. Yet, questions still remained. Chemical analysis of chromosomes showed them to be composed of both protein and DNA. Which substance carried the hereditary information? For many years most scientists favored the hypothesis that protein was the responsible molecule because of its comparative complexity when compared with DNA. After all, DNA is composed of a mere 4 subunits while protein is composed of 20, and DNA molecules are linear while proteins range from linear to multiply branched to globular. It appeared clear that the relatively simple structure of a DNA molecule could not carry all of the genetic information needed to account for the richly varied life in the world around us!
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