Proteomics | SDS PAGE | Applications of Proteomics | Biology Bug

What is Proteomics?

Proteomics is the branch of molecular biology which is the large scale study of proteomes. But what is proteome? A proteome is set of proteins produced in an organism, a system or biological context. Proteome can be studied in specific organism (human, rat) or can be studied in specific organ (liver, pancreas). The proteome is not constant because it differs from cell to cell and changes over a period of time.

Importance of proteome analysis

Proteins are not only the important macromolecules in our body but also the most common effectors of disease pathogenesis and determinants of treatment response. But analysis of proteins is technically challenging and hence we face troubles to develop effective therapeutics. However analysis of DNA and RNA have some role in the prediction of protein function but these results do not always correlate with protein functions. Proteomics help us to overcome these challenges and also in the prediction of protein functions at different levels like post translational modification, domains, motif etc. Despite the inherent limitations of proteomic methodologies, as many as 115 protein based assays have been approved for use by regulatory agencies and commercialize with the great success.

SDS-PAGE or Gel elctrophoresis

Gel based proteomic is the most popular and versatile method of global protein separation and quantification. This is mature approach to screen the protein expression at the large scale. Based on two biochemical characteristics of proteins, two dimensional electrophoresis combines isoelectric focusing which separates proteins according to their isoelectric point and SDS PAGE which separates them further according to their molecular mass. The next typical steps of flow of gel based proteomics are spots visualization and evaluation, expression analysis and finally the protein identification by mass spectrometry. For the study of differentially expressed proteins, two dimensional electrophoresis allows simultaneously to detect, quantify and compare up to thousand protein spots isoform including post translational modifications in the same gel and in a wide range of biological systems.

First dimension

Proteins are amphoteric molecules i.e. they carry both positive and negative charge; hence the net charge on proteins is zero depending upon their amino acid composition. The isoelectric point of protein is the specific pH at which the net charge of protein is zero. Proteins are positively charged at pH values below their isoelectric point and are negatively charged at pH values above their isoelectric point. IEF is an electrophoretic separation based on this specific characteristics of proteins.

Basically the first dimension of two dimensional electrophoresis is achieved with a strip. It is a dry gel that is formed by the polymerization of acrylamide monomers, linked by bis-acrylamide with molecules of covalently linked immobilin, immobilins are chemical components that are derived from  acrylamide and have additional ionizable non amphoteric functions. Immobilins of various pKa can create am immobilized pH gradient inside the gel.

The strip acrylamide gels are dried and cast on a plastic backing. They are rehydrated in a solution containing a pI- corresponding cocktail of carrier ampholytes and with the correct amount of the proteins in the solubilization buffer. The carrier ampholytes are amphoteric molecules with high buffering capacity near their pI.

When an electric field is applied, the negatively charged molecules (proteins and ampholytes) move towards the anode (red electrode) and the positively charged molecules move towards cathode (black electrode). When the proteins are aligned according to their isoelectric point, the global net charge is zero and the protein is unable to move and is then focused. Focusing is achieved with a dedicated apparatus that is able to deliver up to 8000-10000 V, but with a limitation in current intensity to reduce heat.

The equilibration step is critical for 2DE. In this step the strips are with sodium dodecyl sulfate (SDS), an anionic detergent that can denature proteins and form a negatively charged protein complex. The amount of SDS bound to a protein is directly proportional to the mass of protein. Thus, protein that are completely covered by negative charges are separated on the basis of molecular mass.

Second dimension

The SDS denatured and reduced proteins are separated according to an apparent molecular weight, in comparison with a molecular weight maker. Equilibrated strips are embedded with 1% low melting point agarose in TRIS/Glycine/ SDS running buffer and 0.01% bromophenol blue on the top of second dimension gel. When the bromophenol blue migration front reaches the bottom of the gel, the second dimension is finished and the acrylamide gel can be removed from the glass plates.

Application of Proteomics


Expression profiling

Network mapping

Protein modification


It is a process of analysing and identifying all the protein samples. Mining is one of the ultimate excercise in proteomics where one simply resolves proteins to the greatest extent possible. It uses MS with associated database and software tools to identify what exactly is found. The several approaches of mining offer the ability to confirm

Expression profiling

Protein Expression profiling is identification of proteins at different level of stages of organsims or cells eg. Development or disease state. Also to analyze the expression due to some genetic, chemical or physical stimulus eg drug. Expression profiling is actually a specialzed form of mining. It is used especially in the cases where two different stages are compared to see which proteins are expressing differently. This technique is used to detect the potential targets for drug therapy and disease.

Protein network mapping

It is an approach to determine how exactly proteins react with each other in a living system. Protein carry out their function in close association with other proteins. They involve in signal transduction, complex biosynthetic and degradation pathways. Most of the protein protein interaction has been studied in vitro. Then why network mapping is important? Proteomic approaches offer the opportunity to characterize more complex network through creative pairing of affinity capture techniques with analytical proteomics methods. They used to identify the components of multiprotein complexes. Multiple complexes are involved in point to point signal transduction pathways in cells. This technique helps to understand all the components in single pathway.

Protein modifications

Another application of proteomics analysis is to identify how and where the proteins are modified. There are many common post translational modifications which can govern the structure, function and turnover of protein. Also many chemicals, environmental factors or drugs can influence the modification of protein. These modified proteins can be detected with antibiodies but the precise sequence sites of specific modification are not known. Proteomics approaches offer the best  means of establishing both the nature and the sequence of posttranslational modifications. These approaches will provide of chemical modifications in domain.

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How does heat produce in body?-The Biology Bug

This article gives an idea about what exactly heat in your body is? How does it produced and regenerate? What is thermal regulation?

Key points: Endotherms, heat generation in body, organs involved in production of heat, excess production, cooling agents

When you get pimples or boils on your face, sometimes rashes on your body, headache, hair fall  or fatigue, then I am sure you all must have heard by someone in the house saying that there is too much heat in your body. This is absolutely correct because all you’re experiencing are the symptoms of increased heat in your body. But have you ever thought what this heat is? Where does it come from? How is it generated or produced in someone’s body? Further article explains the process of heat generation by various parts in human body.

Let us first understand there are two categories of animals when it comes to thermoregulation. Endotherms which are warm blooded animals and ectotherms that are cold blooded animals. Humans are endotherms which means they have to adjust the body temperature according to the environment unlike the ectotherms which includes fishes, amphibians, reptiles etc. Human body tries to maintain that with the help of several metabolic processes. Heat is one of the byproduct of the metabolic processes which occur simultaneously.

The body finds its own way to release the heat outside and controls thermoregulation. It finds the ways to release excess of heat by skin, acid production or high rate of burning calories. Human body has several sources for production of heat. One of the major sources is liver but it also includes kidneys, muscles and brain. Highest metabolic activity can be seen in the liver. Liver has more than fifty functions to do at regular intervals. The heat generated in the liver is basically due to cellular respiration. Since liver is a large organ, there are many cells that contribute to the heat. Also sometimes when old blood cells are split, they release the bonding energy which again leads to heat production. It is observed that blood leaving from the liver and brain is comparatively warmer than the normal blood.

Now when we talk about various organs in the body which are responsible for the process, it is important to note that there is one organelle which is present in almost every organ of the body which is mitochondrion. This is called as powerhouse of the cell and basic function of this organelle is to produce ATP. Therefore during the production of ATP i.e. energy, some amount of heat is released.     

Another way in which the body produces heat is by hyperthyroidism. Thyroid gland is an endocrine gland which produces certain hormones like T3 and T4. When there is excess production of these hormones, the body starts getting warmer. Thyroid hormonal imbalance can lead to excess heat production. In response to external environment hypothalamus sends a signal to thyroid gland which increases metabolic activity and makes more energy.

What causes excess of heat?

Let us understand one thing that body continuously produces heat to maintain the body temperature and to respond the external environment. But in few cases when the production is not controlled and body can not regular thermal conditions.


There can be multiple reasons for that which include bacteria or any micro-organism which elicits the immune response. In order to kill that organism body increases the temperature. Therefore fever is one of the immune response to react against foreign substance. The only concern in this case is when body goes on increasing the temperature and starts damaging the other cells. Therefore we need to visit the doctor. Similarly we can give an example of mosquito bite. In response to that bite, body produces excess heat in that specific area and it becomes reddish in color.


As we have already discussed that liver is one of the major source for producing heat. It is because all the food i.e. complex substances that we eat goes to the liver, breaks down to the simple substances and then distributed in body. Our liver acts as a checkpoint for all the substances that we eat or drink. Since liver has never encountered medicines at regular basis, it needs to cross check them. It is called as a first pass effect when some amount of medicine is lost due to the liver action. During this effect a lot of metabolic processes occur which contribute to the heat production. Liver is already producing heat via several metabolic processes. In addition to that the medicine causes more heat and the amount of heat depends on the number of medicines. More medicines causes higher metabolic rate and produces lots of heat in the body.    


Generally the food which takes more time to digest or has high amounts of carbohydrates and proteins tend to produce more heat in the body. The body has to spend lot of energy for breaking down the high molecular weight substances. As a results lots of metabolic activity takes place and lead to excess heat production. It also applies to the oily or especially saturated fatty substances. This type of food has more amount of fats but generally body has a tendency to store the fats in adipose tissues and can be useful in starving conditions. But at the same time some of the fats are broken down and involved in metabolic pathways. Hence they also contribute to the heat.

How to reduce heat naturally?

  • According to Ayurveda one should drink plenty amount of water. As we all know that water is a universal solvent and can dissolve almost anything. It also act as a cooling agent.
  • Yogurt and buttermilk are the best option to reduce heat in your body. These substances are slightly acidic in nature and contribute to neutralize the effect of heat.
  • The best way to reduce heat is to consume sweeteners rather than using sugars. Therefore consumption of honey can be benefecial to reduce heat.

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Golgi bodies: Shape, size, structure and function.

Key points: Golgi complex, history, introduction, shape, size, structure, function.


In 1890 Camillo Golgi described apparato reticulate interno i.e. internal reticular apparatus in the nerve cells. In 1900 Holmgren described a system of clear canals, which he called trophospongium. Baker (1951,1953) referred to the Golgi apparatus as lipochodria because of the presumed lipid content. Structure similar to golgi complex have been found in plants. Botanists refer to them as dictyosomes.

What are Golgi bodies?

The Golgi complex is generally a single large structure. Nerve cells, liver cells and most of the plant cells have multiple Golgi complexes, there are about 50 in liver cells. Cells with dispersed Golgi complexes may have hundreds scattered throughout the cytoplasm of the cell.

Shape and size of Golgi complex

The shape is variable in different somatic cell types of animals. Even in the same cells there are variation in different functional stages. It varies from a compact mass to disperse filamentous network. The position of Golgi complex is also variable. They are ectodermal in origin and is polarized between the nucleus and periphery. In exocrine cells it lies between nucleus and secretory pole. The size of Golgi complex is large in nerve and gland cells, but small in muscles cells. The size is linked to the functional state.

Structure of Golgi complex under electron microscope

Electron microscope observations of thin sections reveal the presence of three membranous components:

  1. Flattened sacs or cisternae
  2. Small tubules and vesicles
  3. Large vacuoles and filled with amorphous or granular substance.

These membranous structures are characterized by the absence of ribosomes i.e. they are smooth membranes.

Functions of Golgi complex

There are several functions that have been observed attributed to Golgi complex

  • Role in protein secretion (migration, transport and packaging)

In pancreatic exocrine cells there are distributed proteins (digestive enzymes). Now proteins are formed at ribosomes which are attached to endoplasmic reticulum. The synthesized proteins are then transferred to ER. From there they move towards Golgi apparatus. In the Golgi complex the proteins are contracted and transformed into zymogen granules. These zymogen granules released from Golgi complex and migrate to the surface of the cell. Here the limiting membrane of the zymogen granule fuses with the plasmalemma thus discharging its contents. 

  • Secretion of polysaccharides

Precursors enter the goblet cells from the capillaries of the vascular system. The amino acids are synthesized into proteins on the ribosome of ER. The proteins are then transferred to the cisternae of Golgi complex. The simple sugar molecules go directly from blood stream to cisternae where they are complexed with protein to form a glycoprotein.


In many cells the protein released from the ER is combined with the carbohydrate to produce complex carbohydrates like glycoprotein, mucopolysaccharide, glycogen and glycolipids. Addition of carbohydrate components to the protein occurs in the Golgi complex as well as in ER.

After completion of glycosylation the glycoprotein is released into the lumen of Golgi complex cisternae.

  • Sulphation

Golgi bodies take part in the sulphate metabolism. Compounds containing active Sulphur are formed in two steps process. Sulphate is first activated by ATP in two stages, the process requires two separate enzymes. The process is carried out by enzyme suplotransferases.

  • Plasma membrane formation

Secretory granules originating from Golgi complex fuse with the plasma membrane during the process of exocytosis. The membrane of the granules become incorporated with the plasma membrane and contributes to the renewal of plasma membrane components. Golgi complex plays an important part in the synthesis of carbohydrate components in the plasma membrane.

  • Lipid packaging and secretion

The epithelial cells produce chylomicrons which contains lipids in the form of lipoproteins. The Golgi complex may also be involved in adding of carbohydrates of chylomicrons. Therefore the overall role of Golgi complex is the concentration and modification of secretory material. These changes convert lipid to chylomicrons. The Golgi complex provides the membrane for the envelopment of lipid so that it can be released from the cell.

  • Acrosome formation

In early stages of mammalian development the spermatid of cell has Golgi apparatus which was spherical in shape with parallel flattened cisternae. Later the complex becomes irregular and the cisternae dilate to form sacs. proacrosomic granules appear in the centre of Golgi complex and fuse to form acrosome.


Golgi bodies are ectodermal in origin. They consist of cisternae and large vacuoles. They involve in many processes such as protein synthesis, polysaccharides, lysosomes, acrosome formation etc.

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PROTEINS: Definition its Structures, Domains and Motifs.

Key terms: proteins, structure of proteins, alpha helix, Beta sheet, loops, domains, motifs, folding, unfolding.

I am sure that you all must have heard this term ‘protein’ during your high school or college. But when it comes to study of proteins, sometimes it gets difficult for some people to clearly get that idea and basic concepts behind it. So have you ever imagined how exactly the protein looks like or how does it function at molecular level? Well then this article is for you. Let’s make it simple.

  • What are Proteins?

They are the organic compounds which contains a long chain of amino acids (polypeptide chain) and maintains the structural components of body. They are mainly present in the muscles (eg. myosin), nails, hair (eg. keratin), immune system (eg. antibody) etc. They are also present in the food and dairy products such as egg, milk, pulses, fish etc. These proteins can be extracted and used as supplement or therapeutic purposes.

  • Structure of Proteins

The structure depends on the nature of protein i. e. whether the protein is globular or fibrous in nature. But they are classified in the following four types:

  1. Primary structure
  2. Secondary structure
  3. Tertiary structure
  4. Quaternary structure

1.Primary structure

The primary structure of protein contains a simple chain of amino acids without any loops or turns inside it. They involve formation of peptide bond. The peptide bond is covalent bond formed between carboxyl group and amino group of two amino acids.

2. Secondary structure

This involves the folding of structure with respect to polypeptide chain due to the reaction between atoms. The folding results into two most common structures, alpha helix and beta pleated sheet.

A. alpha helix

The polypeptide chain is coiled spirally in this structure. The backbone forms inner part of the coil while the side chains extend outward from the coil. In this structure the carbonyl group of amino acid is bonded with the hydrogen of amino group via hydrogen bonding. Therefore it gives the appearance of ribbon to the secondary structure.

B. Beta sheet

The linear extended zigzag pleated sheet is formed by the hydrogen bonds which is either intramolecular or intermolecular. The sheet like structure is formed when two or more segments of polypeptide chain are present next to each other and connected via hydrogen bonds. Individual segments in beta sheets are known as beta strands and they are rarely found in proteins because the structure is not stable. When two adjacent beta strands line up, they can form bridges of hydrogen bonds. This stable structure is known as Beta sheet. However there are further two types of beta sheets: a) Parellel and b) Anti-parallel.

a) Parallel beta sheet:When beta strands line up edge to edge in the same direction, it forms highly stable sheet.

b) Anti-parallel beta sheet: Beta strands runs into opposite direction with each other. The anti-parallel conformation is more stable and more common than the parallel one.

  • Loops and turns in the secondary structure
  1. Loops and turns connect alpha helices and beta strands.
  2. The more common cause for a polypeptide chain to contain more loops is to make the structure more compact and stable.
  3. Loops that have only 4-5 amino acid residues are called as turns.
  4. When they have internal hydrogen bonds, loops generally have hydrophillic residues and are found on the surface of the protein. While turns and bends refer to short segments of amino acids that connect the ends of two adjacent segments of anti-parallel beta sheets.

Before moving to the tertiary structure, it is important to understand some basic concepts for the functions of protein such as domains and motifs.

  • Domains, motifs and folds

The polypeptide chains with more than 100 amino acid residues often fold into two or more stable globular units are called domains. The domain is a functional area of protein which performs certain physical or chemical activity. Along with the domains there are certain motifs that represent only the structural characteristics of protein. Motif does not perform functional activity, instead they are a part of domain. In many cases a domain from a large protein still retain to its 3-D structure even if it is separated from polypeptide chain. While it is not in the case of motif because it contains short sequences of amino acids.

3. Tertiary structure

It represents the entire three dimensional conformation of protein. It indicates all the secondary structure helices, loops, turns, bends, sheets and how are they assembled to form a domain in space. It basically explains the 3-D structure of single protein(unlike quaternary structure) and how do all the small components contribute to coiling and compacting the structure to make it stable.

4. Quaternary structure

The quaternary structure of protein involves the clustering of more than one protein chains into a specific shape. This complex structure of protein is formed via various reactions and interactions such as hydrogen bonding, salt bridge formation, disulphide bonds, van der waals forces, covalent bonds etc. It contains many sub units and give rise to a complex structure held by various bonds.