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Saturday, December 31, 2011

Fractionation of Phospholipid by Thin Layer Chromatography (TLC)

Phospholipids are group of lipid molecules containing phosphorus. The bases contain are choline, ethanolamine, inositol and serine phospholipids are frequently found in association with proteins and constitute the essential part of all cell membranes. The phospholipids are amphoteric nature because of the hydrophilic and hydrophobic part and which is of great importance.

Solvent used for Fractionation: chloroform – methanol – acetic acid - water [25:15:4:2]
Other Requirement : Ice cold acetone, Choloroform - Methanol mixture

Procedure: Isolation of phospholipid from egg yolk.
  1. Egg yolk is collected and mixed well, to this 150ml of cold acetone and allowed to stand for 15 mins.
  2. The precipitate is collected by centrifugation. Then the precipitate is extracted with 500ml of chloroform – methanol mixture (2:1) for 3-4 hours at room temperature.
  3. The extract is evaporated to dryness the residue is taken up in a small volume of petroleum ether (25-30 ml)
  4. The phospholipids are re-precipitated  with the addition of cold acetone (200ml)
  5. The precipitate is collected and dried.
Thin Layer chromatography for Phospholipids
  1. The TLC chamber is filled with solvent chloroform – methanol – acetic acid - water [25:15:4:2] and is kept tightly closed.
  2. Dried precipitate is taken in an eppendroff tube and to this petroleum ether is added, Mix well and let it stand
  3. Phospholipid gets dissolved in ether and clear liquid phase is attained.
  4. Transfer this liquid phase to a fresh eppendroff tube.
  5. Using a capillary tube, spot samples on the TLC plate.
  6. When the sample gets dried transfer the plate into TLC chamber for 15 mins (till the solvent moves 3/4th of the plate)
  7. The plates are then taken out and placed in a chamber which is saturated with iodine vapors for about 2 mins.
 Retention Factor (Rf)
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.
Rf = Distance Travelled by compound / Distance Travelled by the solvent

The Rf Values can be calculated from the above equation.

Thin Layer Chromatography (TLC)

This is a technique more or less similar to paper chromatography. In thin layer chromatography (TLC) a thin layer of finely divided substance is deposited on to a flat plate. The sample to be separated is spotted at one end. The plate is then dipped in a glass jar having suitable solvent.

Preparation of Thin Layer:
The plate on to which thin layer is prepared should be uniform and should be thoroughly washed and dried before layer application. Plastic or foil plates can be used. The material of which thin layer is to be made is mixed with water to make a slurry. This slurry is applied on to plate uniformly using plate spreader.

Detection / Development:
There are several methods for detection of compounds in a TLC plate
Spraying the plate with 25 – 50% H2SO4 in ethanol and heating the plate. Spots will show up on the plate.
Iodine vapor is used for organic compounds. The iodine spots disappears rapidly but can be make it permanent by spraying with benzidine solution in absolute alcohol.
Even UV light can be used for detection of certain compounds.

Retention Factor (Rf)
The retention factor, or Rf, is defined as the distance traveled by the compound divided by the distance traveled by the solvent.

Rf = Distance Travelled by compound / Distance Travelled by the solvent

Thin Layer Chromatography Advantages
It is more versatile, Faster and more reproducible.
Often been used to detect drugs, contaminants and adulterants
It has been widely used to resolve plant extracts and many other biochemical preparations.

Friday, December 30, 2011

Principle of Isoelectric Focusing (IEF)

Principle of Isoelectric Focusing (IEF)

Principle of Isoelectric Focusing (IEF)

Isoelectric Focusing or IEF is a method of separating proteins according to their Isoelectric points in a pH gradient. Isoelectric point denoted as pI is defined as the pH at which protein carry no net charge, or pH at which protein become immobile in an electric field.

Isoelectric Point determination

One of the best method for characterizing proteins, because protein separation /purification can be easily done if Isoelectric point of a protein is known.

Isoelectric Focussing Principle

The resolution of Isoelectric Focussing is very high. In normal electrophoretic methods pH between anode and cathode remains constant but in Isoelectric focussing , a pH gradient is arranged.when pH of protein is below its pI proteins become positively charged and it will migrate towards cathode. because of the pH gradient charge of the protein molecule changes while moving forward, there will be a point at which net charge of the protein becomes zero this point is called Isoelectric point. when a protein mixture or a single protein is run under an electric field at specific pH the protein stops moving.

Factors affecting efficiency of Column Chromatography

Factors affecting efficiency of Column Chromatography

Factors affecting efficiency of Column Chromatography

Efficiency of column chromatography or Resolution of column chromatography is dependent on various factors,  some of the few major factors includes

factors affecting chromatography

Column Chromatography 

Dimension of the column:
Columns of various dimensions are available for use, depending upon the process suitable column dimension need to be selected. Height or length / width ratio is important factor, increasing the length of the column shown to have improved separation / resolution. Mostly for desalting purposes long columns are shown to give satisfactory results. But optimization is needed for each process to show effective reproducible results.
Example of certain columns,XK columns are jacketed columns eg: XK 16/40,  XK 50/30, here 50 denotes the inner diameter of the column in millimeters (mm) and 30 is the length or height of the column in centimeters (cms).

Particle size of the matrix:
Particle size also plays important role in the improved separation, In gel filtration chromatography small medium and super fine range particles are available, the particle size and other details will be available on the specification sheet provided during the purchase of the chromatography matrix. Decreasing  the particle size shown to have improved separation.

Solvents used in the column chromatography should not affect the stability of the proteins or it should not cause any deleterious effects, solvents used should be compatible with the column and the matrix used. Generally a solvent with low viscosity is used since the viscosity can affect the flow rate and which in turn affects the separation.

Temperature is also an important factor in chromatography, since proteins can degrade at higher temperatures. Large scale protein purification processes are done mostly at 4oC.lab scale protein purification is usually done at room temperature. Some chromatographic columns can get damaged at higher temperatures of around 60oC. It is advisable to maintain lower temperatures for improving the yield and efficiency of column chromatography.

Flow Rate:
Flow rate can be maintained using a peristaltic pump or  AKTA systems ( Protein purification controller form GE Life Science). For each chromatographic media or matrix the maximum flow rate at which can be used during the process will be given in the specification sheet ( For flow rate conversions click here).
Running the column at Medium flow rate is recommended, because very slow flow rate can lead to zone spreadening and too fast flow rate can cause extensive tailing.

Column Packing (How to Pack a chromatography column) is yet another important factor in the resolution, proper packing of the column is very important to get better results. Few points to remember while column packing
  •  Degas the matrix and buffers, this will help to remove air bubble trapped inside, air bubbles in the column can lead to poor resolution.
  • Pour the matrix / media into the column in a single time attach reservoir to the top of the column.
  • Calculate the amount of matrix required for the column, if your swelling dry matrix calculate amount of dry powder required to get the matrix volume.(click here for matrix volume calculations).
  • Place the column properly, column need to be kept straight.
  • Check the filters (mesh) other tubing connections are proper before starting. Purging the lines (tubing and system lines if using AKTA) can remove air bubbles.
These are the major factor which can affect the resolution or separation in a column chromatography, equilibration, washing, suitable matrix selection etc also can influence the efficiency. process optimization need to be done to get better results. For long term storage, chromatographic matrix can be stored in 20% ethanol. while reusing  3-7 column volumes of water wash can be given to remove ethanol.

Analytical Biochemisrty, Dr. P Asokan
GE Lifescience Chromatography Literature
Tosoh Biosciences, Chromatography Literature
Personal Experience

Matrix or Media used in column chromatography

Column chromatography is technique used to separate compounds of interest from the others using a column finely packed with media/matrix as stationary phase.

Matrix used should have certain properties these includes:
1. Mechanical Stability
2. Chemical Stability
3. Should be chemically inert

Commonly Used Matrices

Cellulose Based :
Eg: Cellufine

Dextran Based :
Eg: Sephadex, Sephacryl

Agarose Based :
Eg: Sepharose, Biogel A

Polyacrylamide Based :
Eg: Biogel - P

Polystyrene Based :
Eg: Dowex - 50

Silica Based :

Distribution Coefficient (Kd) in Gel filtration (GF) / Size Exclusion Chromatography (SEC)

In gel filtration, distribution of particular compound between the inner and outer mobile phase is a function of its molecular size, which is represented by distribution coefficient (Kd).The larger molecules which is usually excluded from the gel beads, such molecules will have a Kd value of zero.Certain molecules which is smaller than the pore size of the gel  beads enters the pores in the gel matrix hence they will have a Kd value of one.For molecules of intermediate size, they will have Kd value between zero and one.This type of variation in the Kd Values makes it possible to separate molecules in the narrow molecular size range.

gel filtration kd value

Thursday, December 22, 2011

Chromatography Calculations: Resolution of Chromatography Peaks,Bed Volume

Chromatography calculations

These are the equations which is used to calculate various things during chromatography including column volume, media requirements for column, resolution etc

Total or Empty column volume

Ve = π * r2 * L or 1/4 π * d2 * L

Ve = empty column volume
r = column radius
L = length of the column (or packed bed)
D = column diameter
If r (or d) and L are expressed in mm, Ve will be in µL. If in cm, then Ve in mL.

Volumetric Flow Rate (VFR) Calculation from Linear Flow Rate (LFR)

This can be useful because the chromatographic media has certain flow rate specification at which it can be operated, for example a chromatographic media specification says use 150 cm/hr flow rate so while setting up the flow rate in peristaltic pump or in other automated system like AKTA volumetric flow rate need to be calculated, using the equation below it can be calculated.

VFR (ml/min) = LFR(cm/hr) / 60 * πr2

we need 150cm/hr and the column used has a diameter of 0.5cm or 0.25cm as radius so,

VFR = 150/60 * 3.14 * (0.25)2 ;

Volumetric flow rate will be ~ 0.5 ml/min

Linear Flow Rate (LFR) Calculation from Volumetric Flow Rate (VFR)

LFR(cm/hr) = VFR(ml/min) * 60 / πr2

Similarly we need 1ml/min and the column used has a diameter of 0.5cm or 0.25cm as radius so,

LFR(cm/hr) = 1 * 60 / ( 3.14 * (0.25)2 )

Linear flow rate will be ~ 300 cm/hr

Bed Volume

Bed volume (L) = Bed height (cm) * Column cross-sectional area (cm2) / 1000

cross-sectional area = π * r2

Packed Bed Volume

Packed bed volume (L) = Column bed height (cm) * cross-sectional area (cm2) * x10-3

cross-sectional area = π * r2

Settled chromatography medium volume

Settled chromatography medium volume (L) = Packed bed volume (L) * compression factor
compression factor can be obtained from the chromatographic media specification sheet.

Slurry volume

Slurry volume (L) = Settled chromatography medium volume (L) / (slurry concentration (%) * x 10-2)

The chromatography medium volume for HIC, IEX, Affinity, Reverse Phase

The chromatography medium volume is calculated from the formula:
The chromatography medium volume (L) =

(Quantity to be bound (g) * (1+ safety margin (%) / 100)) / (Dynamic binding capacity (g/L)

The chromatography medium volume for Gel Filtration Chromatography
The chromatography medium volume (L) =
Sample volume to be processed (L) / (Sample volume in percent of total column volume (%) / 100)

Resolution (Rs)

Chromatographic peak resolution can be calculated using the following equation

Rs = (tR1 – tR2) / 0.5(W1 + W2)

Rs – Resolution

tR1 – Retention time of the first peak

tR2 – Retention time of the second peak

w1 – Width of first peak

w2 – width of second peak

Sources & References

GE Life Sciences - Chromatography medium volume calculators

Tosoh Bioscience LLC: The Chemistry of Innovation - Technical Support – Basic Chromatographic Calculations

Wednesday, December 21, 2011

ELISA Protocol- Types of ELISA- Advantages & Applications of ELISA

ELISA Protocol - Types of ELISA

The Enzyme-Linked Immunosorbent Assay (ELISA) / (EIA) involves coating (binding) of an antigen (Protein) to a solid support such as a membrane (as used in immunoblotting/western blotting) or a 96-well micro plate (ELISA Plate). The coating is done using bicarbonate / carbonate coating buffer. Proteins which have not been separated by electrophoresis can be bound to membranes and analyzed with primary and secondary antibodies as in the immunoblotting procedure. Such analysis are often called dot blots. The more common format is to absorb the antigen to the wells of a 96-well microplate and to use substrates that produce a colored product. The ELISA is suitable for the analysis of large numbers of samples and most of the procedure can be automated.

Antigen Coating
The first step is to bind the antigen to the micro well plate. The antigen used can be a purified protein , peptide or a crude protein extract. The choice of antigen will depend on the application and the nature of the antibodies being tested. Binding to 96-well micro plates is achieved by incubating the wells with a solution containing the antigen. Protein binding to the plate is due to hydrophobic interactions between the protein and the plastic. coating incubation may vary from 2hrs at room temperature or overnight at 4oC.
Once antigen is bound to the plates, it is important to block the remaining surface of the plate / membrane to prevent nonspecific binding and the detection antibodies during subsequent steps. Many Blocking buffers are available to block the free sites on the plate, BSA, non fat dry milk powder etc in PBS(phosphate buffered saline) or TBS (Tris buffered saline) with minute percentage of tween 20 or Triton X-100 can be used as blocking buffer. The protein in the blocking solution will attach to the membrane in all places where the target proteins have not attached. thus when antibody is added which will bind only to the target protein so the background interference will be reduced.
Incubation with Primary & Secondary Antibodies
After blocking the, excess blocking agent is removed by washing the plate / membrane with PBS and tween 20 (wash buffer) for sometime Then the antigen-containing wells are incubated with the primary antibody. Appropriate antibody dilutions need to be made which will better results, Serial dilutions are often carried out and the minimum titer which is giving a good response can be used. Optimization of the antibody concentration need to be done for getting better results. Antibody dilution are made in PBS or TBS. ELISAs are typically carried out in duplicate or triplicate. A positive and a negative control is also included along with the test samples. Primary antibody incubation may vary from 2 hours or even more incubation periods are required to get good results. After incubation with the primary antibody the wells are washed extensively to remove any unbound antibody. Similarly membrane / plate is incubated with 2o antibody with suitable dilutions. incubation period varies from 2 hrs or sometimes even more, secondary antibody will bind to the primary antibody, secondary antibodies are conjugated with enzyme which on reaction with substrate in the developing solution will yield colour. excess antibodies are washed off with wash buffer. there are variety of tags or enzyme labels can be conjugated to the secondary antibody. Most widely used enzymes are Horse Radish Peroxidase(HRP) and Alkaline Phosphatase (AP).
A developing solution having chromogenic substrate is added to the wells, the enzyme reacts with the substrate and gives colour. The intensity of the color will be proportional to the amount of secondary antibody which proportional to the amount of specific primary antibody in the sample. Alternatively a constant amount of a defined antibody can be used to quantify the amount of a specific protein in the sample. ELISA plate readers is basically a spectrophotometer designed to read the individual wells in a 96-well microplate. ELISA plate readers are interfaced with a computer to assist in data management.
most widely used substrates are pNPP (p-nitrophenyl phosphate); BCIP (5-bromo-4-chloro-3-indolyl phosphate); NBT (nitro blue tetrazolium); TMB (3,3',5,5'-tetramethyl-benzidine); DAB (3,3-diaminobenzidine)

Types of ELISA

Elisa Types - Advantages

Indirect ELISA
The method described above is for the indirect ELISA.

Competitive ELISA
Antigen (Sample) is incubated with an unlabelled antibody, these bind each other to form antigen-antibody complex. These bound antibody/antigen complexes are then added to an antigen-coated well. The plate is washed, so that unbound antibody is removed, there will be competition for binding of antigen in the well, then a secondary antibody specific to the primary antibody is added. This secondary antibody is coupled to an enzyme. When a substrate is added, enzyme acts on the chromogenic substrate and emits colour. ELISA reader is used to measure the intensity.

Sandwich ELISA
In sandwich ELISA first the Plate is coated with a capture antibody; to the antibody coated well sample is added and if any antigen present in the sample will binds to capture antibody; an antibody which detects the antigen is added, and this antibody binds to antigen. Finally an enzyme-linked secondary antibody is added, and this secondary antibody binds to detecting antibody when a substrate is added, the secondary antibody having enzyme converts the substrate and colour is developed which can be read in ELISA reader.

Advantages and Applications of ELISA
  1. ELISA can be used to quantify Antibody in the sample
  2. ELISA can be used to quantify Antigen in the sample
  3. Large no of samples can be processed at a time.
  4. Highly sensitive.
ELISA has many applications in invitro disease diagnostics, some of the are
  • Detection of HIV antibodies in serum
  • Diagnosing Lyme Disease
  • Diagnosing Lyme Disease, etc
  • Detecting plant diseases.
  • Application in toxicology for screening of Drugs.
  • Detecting Food Allergens.
Methods in Cell Biology, Mark F. Wiser

Tuesday, December 20, 2011

Interference RNA (RNAi),siRNA Transfection, Antisense RNA Technology methods & applications.

Anti Sense RNA
During mRNA synthesis DNA strand is transcribed to give messenge RNA (mRNA) and this messenger RNA is called as a sense RNA Anti Sense RNA is a short single stranded RNA which can bind the the complementary messenger RNA(mRNA) which is transcribed by the specific gene with in the cell. The binding of the anti sense RNA to the mRNA prevents it from reaching the ribosome for translation.
Antisense RNA Technology
Anti sense RNA drugs are small chemically modified nucleotides which are complementary to its target RNA. Once it is introduced into the cell results in RNA Introduction of antisense RNA into the cell results in the formation of RNA-DNA hybrid, this hybrid will be degraded by RNase H and there by preventing the translation of the that particular mRNA ultimately resulting in gene silencing. Blocking the disease causing protein production has can results in improvement in the treatment of certain disease which otherwise would be difficult to treat using normal methods.
Antisense RNA technology has application in agricultural field also, this technology was used to develop Flavr Savr Tomato which offered improved Flavor and shelf life by delaying the ripening process.

Regulus Therapeutics Inc. is a bio-pharmaceutical company focused on the development of microRNA(miRNA) based drugs targeting fibrosis, metabolism and cardiovascular diseases, cancer, HCV and immune-related diseases.
RNA interference (RNAi) is the process of regulation of genes in the cell. The other names for RNA interference are post translation gene silencing, co-suppression, quelling etc.
RNA interefernce is caused by two types of RNA molecules siRNA – small interference RNA and miRNA – micro RNA. Interference RNA works by targeting RNA induced silencing complex (RISC) to bind and degrade the mRNA.

siRNA Mechanism

Antisense RNA Technology
siRNA Mechanism

Interference RNA (RNAi) Applications
One of the promising application of interference RNA (RNAi) technology is for the treatment of viral infection. stem cells are used as a model for RNAi studies for better understanding of molecular regulations with in the cell.
Study of various gene regulation pathways and associated disease progress.
Si RNA Transfection:
Transfection is the process of introduction of foreign DNA, RNA, or protein molecules into eukaryotic cells. Transfection can be Transient or Stable. In Transient transfection the transfected molecule will only be expressed for a short period of time.
Methods of siRNA Transfection
Physical and chemical methods can be used to transfer DNA/RNA (siRNA) into the cell. Even bacterial and viral methods are also used.
Physical methods involves electroporation, microinjection, sono-poration, impalefection, optical transfection, etc.
Use of calcium phosphate method, liposomes, Polymers etc comes under the chemical means of Transfection.

RNA Interference: Applications in Vertebrates, Frederic Bushman
interference RNA, Wikipedia
Application of RNA interference to study stem cell function: current status and future perspectives,
Gang-Ming Zou1 and Mervin C. Yoder

Saturday, December 17, 2011

Solid State Fermentation / Solid Substrate Fermentation

Solid-state fermentation (SSF) or Solid Substrate Fermentation has been defined as the fermentation process occurring in the absence or near-absence of free water.
SSF employs natural raw materials as carbon source such as cassava, barley, wheat bran, rice bran, sugarcane bagasse, cassava bagasse, various oil cakes (e.g. coconut oil cake, palm kernel cake, soybean cake, ground nut oil cake, etc), fruit pulps (e.g. apple pomace), corn cobs, saw dust, seeds (e.g. tamarind, jack fruit), coffee husk and coffee pulp, tea waste, spent brewing grains, etc

Advantages of Solid State Fermentation

•Process is simple
•Cost Effective
•Less Effluent release, reduces pollution
•High Titers, (High Product yields)
•Aeration Process is easy
•Resembles the natural habitat of some fungi and bacteria
•Easier downstream processing 

Factors which Influence Solid State Fermentation

Selection of Micro-organisms 
This is one of the key factor for improved yields of the product. Bacteria, Yeast and Filamentous Fungi can be used. Filamentous Fungi has shown better results growing in the solid substrate

Substrate also plays important role in determining the growth of micro-organisms, there by increasing the product yield. Substrate is chosen such a way that it should provide physical support as well as nutrients to the growing culture. 

•Substrate is of two types:
One is Specific substrate, which requires suitable value-addition and/or disposal.
The second is for producing a specific product from a suitable substrate.

•Process Optimization 
Includes the optimization of physico-chemical and Biochemical Parameters such as Size, initial moisture, pH and pre-treatment of the substrate, Relative humidity, temperature of incubation, agitation and aeration, age and size of the inoculum, nutrient Supplementation such as N, P and trace elements, supplementation of additional carbon source and inducers, Extraction of product and its purification

Problems Associated with Solid State Fermentation

•Heat Transfer: One of the main difficulty is to control the temperature during the fermentation process.
•Heat is generated during the metabolic activities of micro-organisms, since the substrate used has low thermal conductivity heat removal will be slow.
•When the heat generated goes beyond certain level, which will result in product denaturation and will affect growth of microbe, ultimately ending up in reduction in yield and quality of the product.

Applications of Solid State Fermentation

  • Agro-Food Industry
Traditional Food Fermentations :Koji, Fermented Cheeses

Mushroom Production & spawn Production: Agaricus, Pleurotus

Bioconversion By-products: Sugar pulp Bagasse Composting, Detoxication

 Food Additives: Flavours. Dyestuffs. 
  • Agriculture Industry
 Biocontrol , Bioinsecticide : Beauveria Metarhizium, Tricho derma

PlantGrowth Hormones / Enhancers : Giberellins, Rhizobium, Trichoderma
  • Industrial Fermentation
Enzymes production : Amylases, Cellulases Proteases, Pectinases, Xylanases

Antibiotic production : Pencillin, Feed & Probiotics

Organic acid Production : Citric acid, Fumaric acid,etc

 Fungal Metabolites: Alkaloids

 Ethanol Production: Malting and Brewing


•General and microbiological aspects of solid substrate fermentation
•Basics of SSF
•Pandey, Ashok (2008, June 13). Solid-state fermentation. SciTopics

Thursday, December 15, 2011

Principle, Methods & Factors affecting Lyophilization / Freeze Drying

Lyophilization / Freeze Drying is the process of drying a frozen product by creating conditions for sublimation of ice directly to water vapor. This is achieved in 3 step process, these are

1. Freezing
2. Primary Drying and
3. Secondary Drying


In the first step, the product is frozen solid, which converts the water content of the material to ice. The final temperature must be below the product's eutectic, or collapse temperature, so that it maintains its structural soundness.
Once the product is frozen solid, the condenser and vacuum systems are energized for the next critical process step.

Primary Drying:

In the second stop, the objective is to remove the unbound, or easily removed ice from the product. This water is now in the form of free ice, which is removed by converting it directly from a solid to a vapor, in a process called sublimation. To accomplish sublimation, a uniform source of heat energy is applied to the ice crystals, turning them directly into water vapor.
The product and condenser chambers are placed under vacuum to encourage the orderly migration of water vapor to the system's ice-collecting condenser, and to ensure that the pressure of the water vapor remains below its "triple point", as required for sublimation to occur.
In manifold drying ambient room temperature provides heat to encourage removal of water vapor from frozen samples.

Secondary Drying:

Even after all the free ice is removed by the sublimation process, your product may still contain enough bound water to limit its structural integrity and shelf life.
During secondary drying, the sorbed water, or the water that was bound strongly to the solids in the product, is converted to vapor. This can be a slow process; the remaining bound water has a lower pressure than free liquid at the same temperature, which makes it difficult to remove. Secondary drying actually starts during the primary drying phase, but must be extended after the total removal of the free ice to achieve low enough residual moisture levels.
Freeze-drying is complete when all the free and bound water has been removed, resulting in a residual moisture level that guarantees the desired biological and structural characteristics of the final product.

Methods of Freeze Drying:

Three methods of freeze drying are commonly used:

1. Manifold Drying,
2. Batch Drying, and
3. Bulk drying.

Each method has a specific purpose, and the method used depends on the product and the final configuration desired.

Manifold Method:
In the manifold method, flasks,ampules or vials are individually attached to the ports of a manifold or drying chamber. The product is either frozen in a freezer, by direct submersion in a low temperature bath, or by shell freezing,(Either Liquid Nitrogen or Methanol can be used for freezing) depending on the nature of the product and the volume to be freeze dried.The prefrozen product is quickly attached to the drying chamber or manifold to prevent warming. The vacuum must be created in the product container quickly, and the operator relies on evaporative cooling to maintain the low temperature of the product.

Heat input can be affected by simply exposing the vessels to ambient temperature or via a circulating bath.For some products, where precise temperature control is required, manifold drying may not be suitable.Several vessels can be accommodated on a manifold system allowing drying of different products at the same time, in different sized vessels, with a variety of closure systems. Since the products and their volumes may differ, each vessel can be removed from the manifold separately as its drying is completed. The close proximity to the collector also creates an environment that maximizes drying efficiency.

Batch Method:
In batch drying, large numbers of similar sized vessels containing like products are placed together in a tray dryer. The product is usually prefrozen on the shelf of the tray dryer. Precise control of the product temperature and the amount of heat applied to the product during drying can be maintained. Generally all vials in the batch are treated alike during the drying process, although some variation in the system can occur. Slight differences in heat input from the shelf can be experienced in different areas. Vials located in the front portion of the shelf may be radiantly heated through the clear door. These slight variations can result in small differences in residual moisture.

Bulk Method:
Bulk drying is generally carried out in a tray dryer like batch drying. However, the product is poured into a bulk pan and dried as a single unit.Although the product is spread throughout the entire surface area of the shelf and may be the same thickness as product dried in vials, the lack of empty spaces within the product mass changes the rate of heat input. The heat input is limited primarily to that provided by contact with the shelf. Bulk drying does not lend itself to sealing of product under controlled conditions as does manifold or batch drying. Usually the product is removed from the freeze dry system prior to closure, and then packaged in air tight containers. Bulk drying is generally reserved for stable products that are not highly sensitive to oxygen or moisture.

Factors that affect the efficiency of lyophilization:

There are several factors which can affect the efficency of lyophilization, some of the major factors includes,
  • sample size
  • surface area of the sample
  • thickness of the sample
  • sample characteristics
  • eutectic temperature
  • solute concentration
  • instrument factors
  • condenser temperature
  • vacuum
The larger the surface area of the frozen material, the faster the rate of lyophilization, and, conversely, the thicker the frozen material, the slower the rate of lyophilization. Sample thickness affects the ability of a sample to absorb and transfer heat to the surface undergoing sublimation. Because water vapor must pass through dried material, the rate of lyophilization in thick samples is slower, especially if the dried material collapses onto the surface of the frozen material. Shell freezing minimizes collapsing by increasing the surface area.The volume of the freeze dry flask should be 2 to 3 times that of the material being frozen.

Sources and References:
A Guide to Freeze Drying for the Laboratory, LABCONCO, An Industry Service Publication.

Friday, December 9, 2011

Factors Affecting Protein Stability and Denaturation

Protein Stability

Proteins are often fragile molecules that need to be protected during purification and characterization. Protein denaturation refers to the loss of protein structure due to unfolding. Maintaining biological
activity is often important and protein denaturation should be avoided in those situations. Elevated temperatures, extremes in pH, and changes in chemical or physical environment can all lead to protein denaturation. In general, things that destabilize H-bonding and other forces that contribute to secondary and tertiary protein structure will promote protein denaturation. Different proteins exhibit different degrees of sensitivity to denaturing agents and some proteins can be re-folded to their correct conformations following denaturation. 

Factors Affecting Protein Stability

·         Temperature :

Increase in temperature can disrupt the  hydrogen bonds and other non-polar hydrophobic interactions.
The reason behind this is increased temperature increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.
One of the reason behind the use of heat for sterilization is this, because higher temperature leads to the denaturation of proteins of bacetrial cells there by killing the bacteria.
Example: Protein Coagulation and re-association of egg-white on frying an egg.

Avoiding high temperatures and Keeping protein solutions on ice can reduce temperature induced denaturation.

·         Freeze-thaw :

Freezing and thawing can disturb the native conformation of proteins, pH variation and precipation of buffer components all those can lead to denaturation of proteins.

To avoid freezing induced denaturation determine effects of freezing. Include glycerol in buffers. Store in it will be easy to take out an aliquot and work with instead of freeze thawing the entire solution.

·         Physical denaturation:

Physical denaturation occur due to handling of  proteins, shaking induces aggregation of proteins, vortexing shaking etc can denature proteins very easily

Do not shake, vortex or stir vigorously. (Protein solutions should not foam.), which helps in reducing protein denaturation.

·         Solution effects: To avoid protein denaturation due to the effect of solution, mimicking the  cellular environment: maintaining neutral pH, ionic composition, etc.

·         Dilution effects: dilution effects can be overcome by maintaining protein concentrations > 1 mg/ml as much as possible.

·         Oxidation:

Oxidation of protein can destroy the stability of proteins, to prevent oxidation induced denaturation strong reducing agents such as  DTT (or β-ME) in buffers can added.

·         Heavy metals  :
Heavy metal salts usually contains Hg2+, Pb2+, Ag2+, Ti1+, Cd2+ and other metalls with weigh atomic weights. Since salts are ionic they disrupt salt bridges in proteins, The reaction of heavy metal salts with protein usually leads to an insoluble metal protein salt.

Including EDTA in buffers helps in sequestering the heavy metals there by reducing protein denaturation.

·         Microbial growth:

 To prevent microbial growth sterile solutions can be used or including anti-microbials, and/or freezing helps to prevent microbial growth.

·         Proteases:

Proteases cleaves the protein and thus stability of  the protein will be lost, resulting in denatured proteins, Include protease inhibitors. Keeping protein solutions on ice helps reduce the effect of proteases.

 The optimal conditions for maintaining the stability of each individual protein need to be determined empirically. In general, though, protein solutions should be kept cold (< 4o C) except during assays and other procedures requiring specific temperatures. Many proteins are especially labile and need to be stored at -20oC or -80oC. However, repeated freezing and thawing of protein solutions is often deleterious. Adding 50% glycerol to storage buffers will lower the freezing point and allow storage at -20o. Solutions for working with proteins will often contain heavy-metal chelators and/or antioxidants as protectants.. In addition, proteases may be released during cell disruption and it may therefore be necessary to include protease inhibitors.


Wednesday, December 7, 2011

Screening Cellulase producers using Congo Red clearing zone assay

Cellulose is a polysaccharide which is made up of 100 to 1000 glucose unit linked together by glycosidic bond. Cellulose is the major component in plant cellwall. Cellulose can be obtained from wood pulp and is mainly used to produce paper. it is a renewable source of energy, Cellulose may be hydrolyzed using enzymes to produce glucose, which can be used for the production of ethanol, organic acids and other chemicals. Cellulose is hydrolyzed using enzyme cellulase, which cleaves cellulose into glucose units.

Cellulase enzymes produced chiefly by microbial sources, starting from prokaryotic organisms like bacteria, and protozoans to eukaryotic organisms, that catalyze the cellulolysis. However, there are also the cellulases produced by animal sources and plant materials. Cellulases are inducible enzymes which are synthesized by microorganisms during their growth on cellulosic materials, the screening of cellulase producing microbes can be done using congo red.

Congo Red Clearing Zone Assay

grow isolated microbes, which is needed to be screened for cellulase production in CMC agar, (carboxy methyl cellulose it is soluble form of cellulose) NaNO3, K2HPO4, KCl; MgSO4, yeast extract, glucose. The medium should be solidified using 1.7 % w/v agar., grow culture 2-3 days at 25-30oC and the screening can be done using congo red clearing zone assay.

Congo red clearing zone assay is suitable for qualitative display of cellulase activity. After incubation plates which contain the circular batches of isolated microorganisms are flooded with 0.1% congo red solution and left for 15 min with intermittent shaking. Then destained with 1M NaCl solution. The clearing zone of enzymatic activity will be visible around the batch of growth. The NaCl solution elutes the dye in the clearing zone where the cellulose has been degraded into simple sugars by the enzymatic activity.

List of few cellulase producing microbes

Trichoderma koningii, Trichoderma reesei, Trichoderma viride, Aspergillus niger

Thermoactinomyces sp., Thermomonospora curvata

Cellulomonas sp.


Qualitative Display and Measurement of Enzyme Activity of Isolated Cellulolytic Bacteria

A.S. Ponnambalam1, R.S. Deepthi2, A.R. Ghosh

Research Article, Biotechnol. Bioinf. Bioeng. 2011, Society for Applied Biotechnology.

Cellulase production Kasing Apun, Sarawak, Malaysia, Practical Biotechnology, NCBE

Friday, December 2, 2011

Western Blot / Protein Immunoblot

Western blotting / Immunoblotting is an analytical technique  used to detect a target protein in a sample (containing a complex mixture of proteins) by using a polyclonal or monoclonal antibody specific to that protein. The mixture of protein can be separated based on their size by poly acrylamide gel electrophoresis, The protein bands are transferred to blotting membrane, the most widely used membranes for western blotting are Nitrocellulose and Polyvinylidene fluoride (PVDF) membranes. To get the Comparison between these membranes and applications click here.

Once the gel run is completed, gel is carefully removed from the plate and soaked in western blot transfer buffer, western blot transfer recipe includes tris, glycine and sds and has pH of 8.4 methanol is also added to the transfer buffer. (For every 100ml western blot transfer buffer 20ml methanol is added.)
Membrane Arrangement:  filter papers on top, gel, western blot membrane, filter papers at the bottom.
western blot membrane

Image Source :

Transfer & Staining
Electric current is applied to transfer the protein from the gel to the membrane, transfer time, applied current and voltage need to be optimized to get better results. The blotting membrane can be stained using Ponceau S which is a reversible stain and the dye can be easily removed by washing the membrane with water. Staining with Ponceau S is helpful to know the effectiveness of protein transfer and which doesn't have any deleterious effect on the protein, if the protein is not transferred to the membrane further processing will be waste of time.
The membrane supports used in Western blotting have a high affinity for proteins. Therefore, after the transfer of the proteins from the gel, it is important to block the remaining surface of the membrane to prevent nonspecific binding of the detection antibodies during subsequent steps. Many Blocking buffers are available to block the free sites on the membrane, BSA, non fat dry milk powder etc in PBS(phosphate buffered saline) or TBS (Tris buffered saline) with minute percentage of tween 20 or Triton X-100 can be used as blocking buffer. blocking is done overnight at 4oC. The protein in the blocking solution will attach to the membrane in all places where the target proteins have not attached. thus when antibody is added which will bind only to the target protein so background interference will be reduced. 
Incubation with Primary & Secondary Antibodies
After blocking the membrane overnight, excess blocking agent is removed by washing the membrane with PBS and tween 20 (0.1%) (wash buffer) for sometime, then membrane is incubated in 1o Antibody solution (antibody solution is made in PBS and Tween 20 (0.1%), appropriate antibody dilutions need to be made which will better results, dilutions ranging from 1/50-1/500,000 can be made according to the antibody stock concentration.primary antibodies are not directly detected, tagged secondary antibodies are used for the detection.secondary antibodies are produced after detecting the antibodies belonging to a foreign species within the blood stream of vertebrate Eg: if mouse monoclonal antibodies are used as primary antibody then secondary antibody will be anti mouse IgG obtained from non-mouse host. During incubation with antibody solutions membrane is kept in gel rocker with gentle rocking. 1o antibody incubation is done for 30 mins to 2hrs sometimes more incubation periods are required. Membrane is washed with washing buffer for 5 mins to remove the excess primary antibodies. Similarly membrane is incubated with 2o antibody with suitable dilutions. incubation period varies from 30 mins to 2 hrs, secondary antibody will bind to the primary antibody, secondary antibodies are conjugated with enzyme which on reaction with substrate in the developing solution will yield colour. excess antibodies are washed off with wash buffer. there are variety of tags or enzyme labels can be conjugated to the secondary antibody. Most widely used enzymes are Horse Radish Peroxidase(HRP) and Alkaline Phosphatase (AP).sometimes radioisotopes fluorophores etc are also used, radioisotopes are expensive and has short half-life period.
Developing an immunoblot of  HRP conjugated antibody is explained here, developing solution is made by dissolving the substrate 0.03% diaminobenzidene(DAB) in PBS and 1% CoCl2,   to this 0.1% hydrogen peroxide (H2O2)is added prior to the incubation with the membrane. even preformulated DAB solutions are readily available. Wash the membrane with wash buffer and incubate with developing solution for 30 seconds to one minute,  DAB reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to the target protein. The reaction can be stopped by adding water.
protein band on western blot
Western Blot Membrane Showing Protein Bands

There are various other methods to develop the blot depending on the nature of tag or label used in the secondary antibody.
Important: Touching the membrane with bare hands will give background while developing. Rocking the membrane for sometime with the blocking buffer is shown to give better results. Appropriate dilutions of antibody will yield good results.

Application in Medical Diagnostics

  • HIV western blot test is used as confirmatory test for detecting anti-HIV antibody in human serum sample
  • Western Blotting is also used for confirmatory test for Hepatitis B Infection.
  • Testing Lyme disease

Thursday, December 1, 2011

Bradford Protein Assay

The Bradford protein assay is a colorimetric assay for the determination of protein concentration, It is dependent on the amino acid composition of the measured protein. The Bradford protein assay was developed by Marion M. Bradford. The Bradford protein assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive

The principle is based on the shift in absorbance maximum of acidic solution of coomassie brilliant blue dye from 465nm to 595nm when binding to the protein occurs.The colour of the solution turns from red to blue.This dye specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. It should be noted that the assay primarily responds to arginine residues (eight times as much as the other listed residues) so if you have an arginine rich protein, you may need to find a standard that is arginine rich as well. CBBG binds to these residues in the anionic form, which has an absorbance maximum at 595 nm (blue). The free dye in solution is in the cationic form, which has an absorbance maximum at 470 nm (red). The assay is monitored at 595 nm in a spectrophotometer, and thus measures the CBBG complex with the protein.

A standard protein curve can be made using known concentration of BSA and then the test sample protein concentration can be determined.

Bio-Rad has ready to use bradford protein assay dye reagent, bio-rad assay protocol can be found from the below link