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:
| Characteristics | Compounds | Mixtures |
|---|---|---|
| Composition | Made up of atoms of elements in a fixed proportion | Made up of elements, or compounds, or both in any proportion |
| Nature | Particles are of the same kind | Particles are of different kinds |
| Structure | Always homogeneous | May or may not be homogeneous |
| Appearance | Components cannot be seen separately | Components may or may not be seen separately |
| Preparation | Always involves a chemical change | Involves only physical change |
| Properties | Entirely different from those of the constituents | No property of their own Show the average properties of all the constituents |
| Separation | Components can be separated only by chemical means | Components can be separated by physical means |
| Energy changes | Energy is always evolved or absorbed | Generally no energy is evolved or absorbed |
The following example of iron, sulphur and iron sulphide will make us understand the difference between mixtures and compounds.
| Properties | Iron | Sulphur |
|---|---|---|
| Colour | Greyish black | Yellow |
| Action of magnet | Attracted | Not attracted |
| On stirring the mixture with water | Sinks, and forms the lower layer of iron | Sinks and forms a layer over the iron |
| Action of dilute acids | Dissolves, producing hydrogen | Does not dissolve |
| Action with carbon disulphide | Does not dissolve | Dissolves |
The following experiment shows the difference in properties between the elements iron and sulphur (Fig.4.1).
Read More
Introduction to compounds and elements
Compounds:
The compound is defined as a pure substance containing two or more elements which are combined together in a fixed proportion by mass.
Elements:
An element is the simplest or basic form of a pure substance which cannot be broken into anything simpler than it by physical or chemical methods. The pure substance which is made up of one kind of atoms only. The common examples of elements are hydrogen, carbon, , sulphur, gold etc.
Elements are further classified into three types.
Metals
Non-metals
Semi-metals
There are few elements which possess the characteristics of both metals and non-metals. These are actually border-line elements and are known as semi-metals. Semi-metals are also called as metalloids. A few common examples are: Arsenic, Antimony and Bismuth.
Types of elements based on physical states:
Based on physical states, the elements have been classified as solids, liquids and gases.
Solid elements: Most of the elements are solids at room temperature. For example, copper, silver, gold, potassium, carbon (diamond, graphite), iodine, phosphorous etc.
Liquid elements: Only mercury and bromine exist as liquid at room temperature. Gallium and cesium become liquids at a temperature 302 K and 303 K respectively. These are slightly higher than the room temperature (298 K).
Gaseous elements: Eleven elements exist in the gaseous state at room temperature. These are hydrogen, oxygen, nitrogen, fluorine, chlorine, helium, neon, argon, krypton, xenon and radon.
A compound is also a pure substance like elements. But it represents a combination of two or more elements which are combined chemically.
Types of compounds
The compounds have been classified into two types
Organic compounds
Organic compounds are the compounds which are obtained from living beings (plant and animal). It has been found that all the organic compounds contain carbon as their essential constituent. The organic compounds are quite often known as carbon compounds.
Examples: Methane, ethane, propane, alcohol, etc.
Inorganic compounds
Inorganic compounds have mostly obtained from non-living sources such as rocks and minerals.
Example: Salt, marble, washing soda, baking soda, etc.
Characteristics of compounds:
The halogens are the most reactive elements as a family. Fluorine is the most reactive of all the halogens. The reactivity of the halogens decreases down the group. The high reactivity of halogens is due to the following reasons:
All the halogens have very low dissociation energies. As a result, they can readily dissociate into atoms and react with other substances. As shown below, the dissociation energies of halogens are quite low in comparison to common molecules such as H2, O2 and N2.
Halogens have very high electron affinity values and therefore, have very strong tendency to gain an electron. Thus halogens are very reactive elements due to their low dissociation energies and high electron affinity values. As clear from the values of bond dissociation energies, fluorine has the lowest bond dissociation energy. This is due to weak F-F bond because of the repulsion between the non-bonding electrons in the small molecule. Therefore, it is most reactive among the halogens.
Some of the important chemical reactions of halogens are discussed ahead.
