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
Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a
at a particular place. They are essential tools for
. The enzyme “scans” a DNA molecule, looking for a particular sequence, usually of four to six nucleotides. Once it finds this recognition sequence, it stops and cuts the strands. This is known as enzyme digestion. On double stranded DNA the recognition sequence is on both strands, but runs in opposite directions. This allows the enzyme to cut both strands. Sometimes the cut is blunt, sometimes the cut is uneven with dangling nucleotides on one of the two strands. This uneven cut is known as sticky ends.
A blunt end may look like this:
A sticky end like this:
Most plasmids used for recombinant technology have recognition sequences for a number of restriction enzymes. This allows a scientist to choose from a number of places to cut the plasmid with a restriction enzyme. Ligation enzymes can then be used to sort of paste in new genomic sequences. These mutated, or recombined, plasmids can then be grown up in bacterial cells and used for a number of purposes, including the addition of genes to mammalian genomes.
You always want to read carefully the information sheet that comes with your enzymes as well as the catalogue information. The better you know your enzyme, the more likely you will be to have a successful digestion. Most enzymes come in glycerol solution as a storage buffer, but enzymes don’t work well in the presence of high glycerol concentration. You want to be sure to dilute the glycerol content down to less than 5% to ensure proper enzymatic activity.
Problems with enzyme activity can occur under the following conditions:
Some other helpful tips for working with enzymes include:
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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(Nutrition Comes Alive, The Nutrient Connection Developed by the Division of Nutritional Sciences Extension Service, Cornell University, 1986)
All foods are ready-to-eat. Values taken from Nutritive Values of Foods, USDA Bulletin.
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All amino acids have a simple chemical backbone with an amine group (the nitrogen containing part) at one end. At the other end is the acid part. This backbone is the same for all amino acids. The difference between them depends on a distinctive structure, the chemical side chain, that is attached to the backbone. It is the nature of the side chain that gives identity and chemical nature to each amino acid. Twenty amino acids with 20 different side chains make up the proteins of all living tissue.
The amino acids that make up proteins differ from fats and carbohydrates in that they contain the element nitrogen. Proteins differ from each other in the sequence of the amino acids that form a particular chain. They also differ in the way that the protein chain (also called a peptide chain) is linked, coiled, or twisted. (See AMINO ACIDS Transparencies #1 and #2 in Resources.)
Chemically, the backbone of every chain is -C-C-N-. This backbone is also called a peptide chain. If two amino acids join in a chain, it is called a dipeptide. A number of amino acids in a chain are called polypeptide. Molecules of water bind to both the backbone and polar groups of proteins. Polypeptide and proteins are formed from amino acids by a condensation reaction in which one amino acid loses -OH from -COOH and another loses -H from -NH2 to form a peptide bond. Repetition of this reaction (polymerization) converts dipeptide to polypeptide and these in turn to proteins. A strand formula for an amino acid, with the variable group R, has been used in the diagram. Breakdown of proteins to polypeptide to amino acids is the reverse process, an enzyme-catalyzed hydrolysis. (See POLYPEPTIDES AND PROTEINS transparency in Resources.)
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