Isolation, Purification and Characterization of RuBisCo at

different stages of Spinach leaves( Spinacia oleracea )

PROJECT REPORT (2014-15)

Submitted To

University Institute of Engineering and Technology

Panjab University, Chandigarh

In the partial fulfillment for the award of degree

Of

BACHELOR OF ENGINEERING

In

BIOTECHNOLOGY

Project Supervisor: Submitted by:

Dr.Parminder Kaur Anshuli Khanna (UE111008)

Ayushi Sharma (UE111012)

Garima Bansal (UE111015)

Ishita Bansal (UE111022)

Mitali Arora (UE111040)

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CONTENT

Introduction………………………………………

Preparation of Solutions…………………………

Preparation of Spinach Leaf Extract…………..

Ammonium Sulfate Precipitation……………….

Dialysis………………………………………………………

Barium Chloride Test……………………………………

Size Exclusion Chromatography………………………

Using Sephadex G25

Using Sepharose 6B

Lowry Protein Assay…………………………………….

SDS Page Elecrophoresis……………………………….

Procedure involved………………………………………

References……………………………………………………

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ACKNOWLEDGEMENT First of all we bow in reverences to the almighty for blessing us with strong will power, patience and confidence, which helped us in the completing the present work.We would like to express my sincere thanks to Dr. Parminder Kaur for giving us an excellent guidance and constant encouragement. We respectfully acknowledge our profound sense of gratitude and heartfelt appreciation to her for giving opportunity to join her esteemed group and for moral support , generosity encouragement, benevolence that has bestowed upon us without have been impossible to complete this project.We are equally grateful to Dr. Sanjeev Puri, HOD, Department of Biotecnology, University Institute Of Engineering And Technology (UIET),Chandigarh ; for his help and invaluable guidance provided during the studies.With generous perception of moral obligation, we acknowledge our reverences and gratitude to our guides Mr. Sukhpal , Mrs. Ramneek, Mr. Naveen and Mr. Arun Raina. We thank them for accomplishing us in executing the project and being very critical during scientific discussion and very humble as well. It helped us to improve and excel every time.Our acknowledgment will be incomplete if we do not mention our family. We pay our gratitude to our parents for their blessing which helped us to achieve goal successfully. There are no words to express our feelings toward them.

.Anshuli khanna,Ayushi Sharma, Garima Bansal,Mitali Arora,

CERTIFICATE

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This is to certify that:

Anshuli khanna (UE111008),Ayushi Sharma(UE111012), Garima Bansal(UE111015),Mitali Arora(UE111040),

Are student of BACHELOR OF ENGINEERING in BIOTECHNOLOGY in University Institute Of Engineering And Technology (UIET), Panjab University ,Chandigarh . they have been sincerely and painstakingly working for the execution of the project entitled:-“ Isolation, Purification and Characterization of RuBisCo at different stages of SPINACH leaves(Spinacia oleracea)”There work is authentic and not been borrowed, copied or submitted anywhere else for the fulfillment of any other degree. These students are diligent , honest and committed to their project whole heartedly and capable of working in any team.

DR. PARMINDER KAUR (Project Supervisor)

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Introduction

RUBISCORibulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviation RuBisCO, is anenzyme involved in the first major step of carbon fixation, a process by which atmospheric carbon dioxide is converted by plants to energy-rich molecules such as glucose.  RuBisCO is the most abundant protein in leaves, accounting for 50% of soluble leaf protein inC3 plants (20–30% of total leaf nitrogen) and 30% of soluble leaf protein in C4 plants (5–9% of total leaf nitrogen).

Properties :

1. RuBisCO is important biologically because it catalyzes the primary chemical reaction by which inorganic carbon enters the biosphere. While many autotrophic bacteria and archaea fix carbon via the reductive acetyl CoA pathway, the 3-hydroxypropionate cycle, or the reverse Krebs cycle, these pathways are relatively smaller contributors to global carbon fixation than that catalyzed by RuBisCO.

2.  Phosphoenolpyruvate carboxylase, unlike RuBisCO, only temporarily fixes carbon.3. Reflecting its importance, RuBisCO is the most abundant protein in leaves, accounting

for 50% of soluble leaf protein in C3  plants  (20–30% of total leaf nitrogen) and 30% of soluble leaf protein in C4  plants  (5–9% of total leaf nitrogen).

4. RuBisCO is usually only active during the day as ribulose 1,5-bisphosphate is not regenerated in the dark.

5. Upon illumination of the chloroplasts, the pH of the stroma rises from 7.0 to 8.0 because of the proton (hydrogen ion, H+) gradient created across the thylakoid membrane.[14] At the same time, magnesium ions (Mg2+) move out of the thylakoids, increasing the concentration of magnesium in the stroma of the chloroplasts.RuBisCO has a high optimal pH (can be >9.0, depending on the magnesium ion concentration) and, thus, becomes "activated" by the addition of carbon dioxide and magnesium to the active sites 

RuBisCo Turnover:

Despite its huge importance in life , RuBisCo is, by enzyme standards, rather slow, which a catalytic turnover rate of between 3 and 10 molecules per second.

NIZO researchers have developed a technology to make the most abundant plant protein in the world available for food applications while maintaining its nutritional and functional properties.

RuBisCo, the most abundant protein in the world, present in every “green” plant can now be extracted as a protein ingredient for the food market. NIZO food research has developed an extraction method resulting in a colorless protein isolate having an excellent solubility.

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RuBisCo combines good nutritional properties with a good techno-functional performance.

With an ever growing world population and increasing demand for high nutrition foods, there is an enormous pressure on the food production system to fulfill this demand while keeping the environmental impact as low as possible. Plant proteins are known to be more sustainable than animal proteins and more cost effective. (Partly) replacing animal protein in existing products with (new) plant protein ingredients or developing new plant protein based products may contribute to an efficient use of available proteins.

Ribulose-1,5-bisphosphate carboxylase oxygenase, most commonly known by the shorter name RuBisCO, is an enzyme that catalyzes the first major step of carbon fixation, a process by which atmospheric carbon dioxide and water is converted to energy-rich molecules such as glucose, using sunlight. In green parts of plants, the protein RuBisCo can make up to 50% of total amount of the protein fraction.

NIZO food research has filed a patent application for the extraction process of RuBisCo from green plants that results in a protein ingredient that has maintained its techno-functional properties, such as solubility and gelling behavior.

Extraction buffer

Buffer systemThe first choice we have to make is that of the nature and the pH of the buffer system we want to use. This depends on: the stability of the target protein with respect to pH and the bufferring compound. the purification procedure. To avoid time and protein loss caused by an additional buffer

exchange step, it is advisable to choose a buffer that is compatible with the first chromatography step (see chromatography).

AdditivesDepending on the target protein, it may be necessary to add compounds to the lysis buffer:

to improve the stability of the target protein.  to keep the protein in solution.

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Metal chelators

EDTA, EGTA

reduce oxidation damage, chelate metal ions

Salts NaCl, KCl, (NH4)2SO4

maintain ionic strength of medium

Reducing agents

DTT, DTE ,Mercaptoethanol

reduce oxidation damage

PROTEIN PRECIPITATION

Protein solubilityThe solubility of proteins in aqueous buffers depends on the distribution of hydrophilic and hydrophobic amino acid residues on the protein’s surface. Hydrophobic residues predominantly occur in the globular protein core, but some exist in patches on the surface. Proteins that have high hydrophobic amino acid content on the surface have low solubility in an aqueous solvent. Charged and polar surface residues interact with ionic groups in the solvent and increase the solubility of a protein. Knowledge of a protein's amino acid composition will aid in determining an ideal precipitation solvent and methods.Important Notes:

Precipitation has an advantage over dialysis or desalting methods in that it enables concentration of the protein sample as well as purification from undesirable substances.

One disadvantage of protein precipitation is that proteins may be denatured, making the pellet difficult to re-solubilize.

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A single precipitation may not be sufficient to remove all types and concentrations of interfering contaminants. In such cases, repeated precipitation may be performed. However, because some sample loss will accompany each cycle of precipitation, use only the number of cycles necessary for the application.

Ammonium sulfate precipitationAmmonium Sulfate Precipitation is a classic first step to fractionate proteins by causing perturbations in the solvent with respect to ionic strength. Historically, separation methods were limited and as a result precipitation methods were highly used with very fine cuts in precipitation conditions. As more choices of inexpensive and quality resins are commercially available precipitation steps are typically limited to one or two initial cuts in the beginning of purification or simply used to concentrate the proteins.

PRINCIPLEAmmonium sulfate precipitation is a method used to purify proteins by altering their solubility. It is a specific case of a more general technique known as salting out.Ammonium sulfate is commonly used as its solubility is so high that salt solutions with high ionic strength are allowed.The solubility of proteins varies according to the ionic strength of the solution, and hence according to the salt concentration. Two distinct effects are observed:

1. at low salt concentrations, the solubility of the protein increases with increasing salt concentration (i.e. increasing ionic strength), an effect termed salting in.

2. As the salt concentration (ionic strength) is increased further, the solubility of the protein begins to decrease. At sufficiently high ionic strength, the protein will be almost completely precipitated from the solution (salting out).

Since proteins differ markedly in their solubilities at high ionic strength, salting-out is a very useful procedure to assist in the purification of a given protein.

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The commonly used salt is ammonium sulfate, as1. it is very water soluble, 2. forms two ions high in the Hofmeister series, and 3. has no adverse effects upon enzyme activity. It is generally used as a saturated aqueous solution which is diluted to the required concentration, expressed as a percentage concentration of the saturated solution (a 100% solution).

ADVANTAGES1. This technique is useful to quickly remove large amounts of contaminant proteins, as a

first step in many purification schemes. 2. It is also often employed during the later stages of purification to concentrate protein

from dilute solution following procedures such as gel filtration.3. it easily causes the reversible precipitation of the protein and4. is non-denaturing to the protein structure.

Points to Consider For Ammonium Sulfate Precipitation. Addition of solid – Add the solid slowly. Simply dumping in the salt at one time will

cause the initial concentration to be much higher as the solid dissolves, resulting in the wrong protein to be precipitated. Add the solid 1⁄4 at a time while stirring on a stir plate. Conducting this in the cold room. Avoid frothing of your solution, this indicates denatured protein at the water-air interface.

Tables of Ammonium Sulfate Addition – There are tables available to tables to use for fine-tuning your ammonium sulfate precipitations.

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Dialysis Methods for Protein

In working with proteins and nucleic acids, it is often necessary to eliminate small molecular weight substances such as reducing agents [dithiothreitol (DTT), 2-mercaptoethanol (BME)], non-reacted crosslinking or labeling reagents (sulfo-SMCC, biotin) or preservatives (sodium azide, thimerosol) that might interfere with a subsequent step in the experimental procedure. Similarly, it is often desirable to exchange the protein sample into a different buffer system for downstream application such as electrophoresis, ion exchange or affinity chromatography. Dialysis is one method for accomplishing both contaminant removal and buffer exchange for macromolecular samples such as proteins.

PRINCIPLEDialysis is a separation technique that facilitates the removal of small, unwanted compounds from macromolecules in solution by selective and passive diffusion through a semi-permeable membrane. A sample and a buffer solution (called the dialysate, usually 200 to 500 times the volume of the sample) are placed on opposite sides of the membrane. Sample molecules that are larger than the membrane-pores are retained on the sample side of the membrane, but small molecules and buffer salts pass freely through the membrane, reducing the concentration of those molecules in the sample. Changing the dialysate buffer removes the small molecules that are no longer in the sample and allows more contaminants to diffuse into the dialysate. In this way, the concentration of small contaminants within the sample can be decreased to acceptable or negligible levels.

Dialysis works by diffusion, a process that results from the thermal, random movement of molecules in solution and leads to the net movement from areas of higher to lower concentration (until an equilibrium is reached). In dialysis, unwanted molecules inside a sample-chamber diffuse through a semi-permeable membrane into a second chamber of liquid or dialysate.

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Because large molecules can not pass through the pores of the membrane, they will remain in the sample chamber. By contrast, the small molecules will freely diffuse across the membrane and obtain equilibrium across the entire solution volume, effectively reducing the concentration of those small molecules within the sample.

Protein Concentration using Dialysis Tubing

Many samples will take on water or buffer during the dialysis process due to osmotic pressure. This occurs frequently with samples that have a high starting salt concentration or if a component of the sample is hygroscopic. In the case of high starting salt concentration, osmosis causes water to enter the sample faster than buffer salts within the sample are able to diffuse out, resulting in the swelling of the sample within the dialysis sample compartment. When this occurs, it may be desirable to return the sample to its original concentration, or to decrease the sample volume even further.To concentrate the sample, dialysis membrane containing the sample is placed in a small plastic bag containing a solution of hygroscopic compound instead of ordinary dialysate. To avoid contamination of the sample, the hygroscopic compound must be composed of molecules that are larger than the pore size of the dialysis tubing (e.g., high-molecular weight polyethylene glycol). With this set-up, concentration occurs upon diffusion of the water (osmosis) and other small molecules out of the sample and into the hygroscopic solution.Another method to concentrate samples is through forced dialysis. Vacuum is applied to a sample contained within a dialysis membrane; this effectively "pulls" water, buffer salts and other low-MW compounds out of the dialysis sample-chamber. Another form of diafiltration involves "pushing" samples through a dialysis membrane by centrifugal force; this is the basis for protein concentrators, which have become popular in recent years.

BARIUM CHLORIDE TEST You can test to see if a solution contains sulfate ions by using barium chloride. If barium chloride solution is added to a sample of water containing sulfate ions, barium sulfate is formed. Barium sulfate is insoluble in water, and will be seen as a white precipitate.The test is done in the presence of dilute hydrochloric acid to remove any carbonate or sulfite ions which may be present. These ions will also produce a precipitate which would confuse the results. Barium chloride is readily soluble in water and is toxic.

Principle:Barium chloride test is based on the reaction of soluble sulphate with barium chloride in presence of dilute hydrochloric acid to form barium sulphate which appears as solid particles (turbidity) in the solution.

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Barium sulphate reagent contains barium chloride, sulphate free alcohol and small amount of potassium sulphate.

Observation:The turbidity produce in sample solution should not be greater than standard solution. If turbidity produces in sample solution is less than the standard solution, the sample will pass the limit test of sulphate and vice versa.

Reasons:Hydrochloric acid helps to make solution acidic.Potassium sulphate is used to increase the sensitivity of the test by giving ionic concentration in the reagentAlcohol helps to prevent super saturation.

Size-exclusion chromatographySize-exclusion chromatography (SEC) is a chromatographic method in which molecules in solution are separated by their size, and in some cases molecular weight. It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. Typically, when an aqueous solution is used to transport the sample through the column, the technique is known as gel-filtration chromatography, versus the name gel permeation chromatography, which is used when an organic solvent is used as a mobile phase. SEC is a widely used polymer characterization method because of its ability to provide good molar mass distribution (Mw) results for polymers. Size exclusion chromatography is used for semi-preparative purifications and various analytical assays. It is a separation technique which takes the advantage of the difference in size and geometry of the molecules.

Principle:

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Size exclusion chromatography (SEC) is the separation of mixtures based on the molecular size (more correctly, their hydrodynamic volume) of the components. Separation is achieved by the differential exclusion or inclusion of solutes as they pass through stationary phase consisting of heteroporous (pores of different sizes) cross linked polymeric gels or beads. The process is based upon different permeation rates of each solute molecule into the interior of gel particles. Size exclusion chromatography involves gentle interaction with the sample, enabling high retention of biomolecular activity. For the separation of biomolecules in aqueous systems, SEC is referred to as gel filtration chromatography (GFC), while the separation of organic polymers in non-aqueous systems is called gel permeation chromatography (GPC). The basic principle of size exclusion chromatography is quite simple. A column of gel particles or porous matrix is in equilibrium with a suitable mobile phase for the molecules to be separated. Large molecules are completely excluded from the pores will pass through the space in between the gel particles or matrix and will come first in the effluent. Smaller molecules will get distributed in between the mobile phase of in and outside the molecular sieve and will then pass through the column at a slower rate, hence appear later in effluent

Applications Purification. Desalting. Protein-ligand binding studies. Protein folding studies. Concentration of sample. Copolymerisation studies. Relative molecular mass determination

. Advantages

1. The advantages of this method include good separation of large molecules from the small molecules with a minimal volume of eluate, and that various solutions can be applied without , all while preserving the biological activity of the particles to be separated.

2. The technique is generally combined with others that further separate molecules by other characteristics, such as acidity, basicity, charge, and affinity for certain compounds.

3. With size exclusion chromatography, there are short and well-defined separation times and narrow bands, which lead to good sensitivity.

4. There is also no sample loss because solutes do not interact with the stationary phase.

Disadvantages 1. only a limited number of bands can be accommodated because the time scale of the

chromatogram is short, and, in general, there has to be a 10% difference in molecular mass to have a good resolution.

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sephadex G-25 This media is an economic gel filtration media based on cross-linked dextran. The hydrophilic matrix minimizes nonspecific adsorption and gives high recoveries during desalting and buffer exchange of proteins and nucleic acids.

Bead structure Sephadex is a bead-formed gel prepared by cross- linking dextran with epichlorohydrin. It is supplied in its dry form. The gel swells in aqueous solutions Different types of Sephadex differ in their degree of cross-linking and hence in their degree of swelling and their molecular fractionation range. Sephadex G-25 is one of eight different G-types ranging from G-10 for small molecules to G-200 for large molecules.

Separation principle Gel filtration separates molecules according to their relative sizes. In Sephadex, the degree of cross- linking of the dextran determines the extent to which macromolecules can permeate the beads. Large molecules are totally excluded while smaller sized molecules enter the beads to varying extents according to their different sizes. Large molecules thus leave the column first followed by smaller molecules in the order of their decreasing size. Sephadex G-25 has a fractionation range for globular proteins of 1000–5000 molecular weight. The separation range of Sephadex G-25 makes it suitable for group separation work such as the removal of low molecular weight contaminants from molecules larger than about 5000 molecular weight.

Stability The mechanical strength and pH stability of Sephadex gel filtration media depend on the degree of cross-linking. Sephadex G-25 is one of the more rigid of the family and has a working pH range of 2–13. It may be safely stored in 0.01 M NaOH without affecting its performance. 20% ethanol may also be used for storage. For cleaning-in-place and sanitization, 60–90 minutes exposure to 0.2M NaOH followed by flushing with water or buffer is recommended. This procedure can be used for hundreds of cleaning cycles. Sephadex media can be autoclaved in their wet form (pH 7.0) at 120 °C for 30 minutes. The rigidity of the matrix means that Sephadex G-25 can be used at relatively high flow rates for rapid separations. Please refer to the applications section for details.

Cleaning a packed column When a column has been in use for some time, it may be necessary to remove precipitated proteins or other contaminants that have built up on the gel bed. Columns packed with Sephadex

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G-25 may be cleaned with 2 column volumes of 0.2 M NaOH or a non-ionic detergent solution (60–90 minutes exposure). The frequency of cleaning will depend on the nature of the sample material and should be worked out on a case-by-case basis. Gel Filtration using Sepharose 6BSepharose is a beaded agarose gel filtration medium with a broad fractionation range. Three different agarose contents are available: 2%,4% and 6%, designated 2B, 4B and 6B, respectively. As agarose concentration increases porosity decreases, thus increasing rigidity and altering the fractionation range; nucleic acids and polysaccharides with molecular weights up to -4X107 can be separated on Sepharose 2B.

Broad fractionation range- High exclusion limits- Negligible non-specific adsorption- Appearance: white suspensionSepharose melts upon heating to 40EC, cannot be autoclaved, and the bead structure may be damaged upon freezing. Due to the presence of 3,6-anhydro-L-galactose, the matrix is resistant to biological degradation. Sepharose is stable in aqueous (including saline) solutions at pH 4-9. Use of dissociation media such as guanidine hydrochloride and urea, chaotropic salts such as KSCN, and oxidizing agents is not advisable because these reagents may disrupt the hydrogen bonds which stabilize the matrix.Usage and RegenerationSepharose are supplied pre-swollen as suspensions in distilled water. Before packing a column, dilute the required amount of gel with starting buffer to form a thick slurry, about 75% of which is settled resin, then degas the slurry. Pass 2-3 column volumes (CV) of eluent through the gel to equilibrate the bed. Sepharose contains a small number of ionic sulfate and carboxyl groups which may cause adsorption of basic proteins at low ionic strengths. Therefore, eluents with ionic strengthsexceeding 0.02 M are sometimes necessary. The gels can be cleaned as indicated below and stored at 4-8EC in a suitable antimicrobial agent (e.g., 20% ethanol) for indefinite time periods. Sepharose should be cleaned in the column or batchwise with a non-ionic detergent solution.

ESTIMATION OF PROTEIN BY LOWRY’S METHOD

PRINCIPLE:The principle behind the Lowry method of determining proteinconcentrations lies in the reactivity of the peptide nitrogen[s] with the copper[II] ions under alkaline conditions and the subsequent reduction of the FolinCiocalteay phosphomolybdic phosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of

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aromatic acids . The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained at 10 - 10.5.The Lowry method is sensitive to low concentrations of protein. The concentrations ranging from 0.10 - 2 mg of protein per ml to concentrations of 0.005 - 0.10 mg of protein per ml.major disadvantage of the Lowry method The major disadvantage of the Lowry method the narrow pH range within which it is accurate. However, we will be using very small volumes of sample, which will have little or no effect on pH of the reaction mixture.A variety of compounds will interfere with the Lowry procedure. These include some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents .The ammonium ions, zwitter ionic buffers, nonionic buffers and thiol compounds may also interfere with the Lowry reaction. These substances should be removed or diluted before running Lowry assays.SDS PAGESDS-PAGE, with full name of sodium dodecyl sulfate polyacrylamide gel electrophores, is the most widely used technique to separate proteins from complicated samples of mixture, plays key roles in molecular biology and wide range of subfield of biological research. Being present a electricity, proteins migerate towards the negative anode inside the poly-acrylamide gel under denaturing conditions. In SDS-PAGE, the detergent SDS and a heating step determine that the electrophoretic mobility of a single kind of protein is only affected by its molecular weight in the porous acrylamide gel.SDS–polyacrylamide gel electrophoresis (SDS–PAGE) is the most widely used methodfor analysing protein mixtures qualitatively. It is particularly useful for monitoringprotein purification and, because the method is based on the separation of proteinsaccording to size. Samples to be run on SDS–PAGE are firstly boiled for 5 min in sample buffer containingb-mercaptoethanol and SDS. The mercaptoethanol reduces any disulphide bridgespresent that are holding together the protein tertiary structure, and the SDS bindsstrongly to, and denatures, the protein. Each protein in the mixture is therefore fullydenatured by this treatment and opens up into a rod-shaped structure with a series ofnegatively charged SDS molecules along the polypeptide chain. On average, one SDSmolecule binds for every two amino acid residues. The original native charge on themolecule is therefore completely swamped by the negatively charged SDS molecules.The rod-like structure remains, as any rotation that tends to fold up the protein chainwould result in repulsion between negative charges on different parts of the proteinchain, returning the conformation back to the rod shape. Once thesamples are all loaded, a current is passed through the gel. The samples to be separatedare not in fact loaded directly into the main separating gel. When the main separatinggel (normally about 5 cm long) has been poured between the glass plates and allowed

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to set, a shorter (approximately 0.8 cm) stacking gel is poured on top of the separatinggel and it is into this gel that the wells are formed and the proteins loaded. The purposeof this stacking gel is to concentrate the protein sample into a sharp band before itenters the main separating gel. However, as they pass through the separating gel the proteins separate,owing to the molecular sieving properties of the gel. Quite simply, the smaller theprotein the more easily it can pass through the pores of the gel, whereas large proteinsare successively retarded by frictional resistance due to the sieving effect of the gels.Being a small molecule, the bromophenol blue dye is totally unretarded and thereforeindicates the electrophoresis front. When the dye reaches the bottom of the gel, thecurrent is turned off, and the gel is removed from between the glass plates and shakenin an appropriate stain solution and then washed in destain solution. The destain solution removes unbound background dye from the gel, leaving stained proteins visible as blue bands on a clear background. A typical minigel would take about 1 h to prepare and set, 40 min to runat 200 V and have a 1 h staining time with Coomassie Brilliant Blue. Upon destaining,strong protein bands would be seen in the gel within 1020 min, but overnightdestaining is needed to completely remove all background stain. Vertical slab gelsare invariably run, since this allows up to 10 different samples to be loaded onto asingle gel.

Various factors affect the properties of the resulting gel.Higher concentration of ammonium persulfate and TEMED will lead to a faster gelation, on the other hand, a lower stability and elasticity.The optical temperature for gel gelation is 23°C-25°C. Low temperature will lead to turbid, porous and inelastic gels.The pH is better to be neutral and the gelation time shoud be limited in 20-30 min.

Materials and requirements 50mM Tris-Hcl pH 8.0 50mM Nacl 1mM EDTa

o gm PVP (polyvinypyrollidone) 70 microliter mercaptoethanol Final volume of extraction buffer was 80ml for 20 gm spinach. Ammonium sulphate salt Acetone Ethanol Dialysis membrane 12-14 Kda

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Sephadex G-25 Barium chloride Reagents

o 2% Na2CO3 in 0.1 N NaOHo 1% NaK Tartrate in H2Oo 0.5% CuSO4.5 H2O in H2Oo Reagent I: 48 ml of A, 1 ml of B, 1 ml Co Reagent II- 1 part Folin-Phenol [2 N]: 1 part watero BSA stock (20mg/100ml)

Polyacrylamide gel electrophoresis

Buffer A

1.5M Tris.HCl (pH 8.9)

Buffer B

0.5M Tris.HCl (pH 6.8)

Acrylamide stock

Acrylamide 30g

Bis-acrylamide 0.8g

Final volume was made to 100ml with double distilled water.

Ammonium persulphate (APS): 10% (Prepared fresh)

Separating gel (Prepared fresh)

Acrylamide stock 1.7ml

Double distilled water 1.9ml

Buffer A 1.3ml

APS (10%) 50l

SDS (10%) 50l

TEMED 2l

Stack gel (Prepared fresh)

Acrylamide stock 330l

Double distilled water 1.4ml

Buffer B 250l

APS (10%) 20l

SDS (10%) 20l

TEMED 2l

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Sample buffer (2X) (Prepared fresh)

SDS (40%) 100l

Beta-mercaptoethanol (–Me) 40l

0.5M Tris-HCl (pH 6.8) 100l

Glycerol 100l

Bromophenol blue (BPB) (33 mg%) 160l

Running Buffer (Prepared fresh)

Tris 3g

Glycine 14.4g

SDS (10%) 1ml

Final volume was made to 1000ml with double distilled water.

Comassie blue stain

Methanol 250ml

Glacial acetic acid 50ml

Comassie blue (R250) 250mg

Final volume was made to 500ml with double distilled water.

Destaining solution

Methanol 62.5ml

Glacial acetic acid 17.5ml

Final volume was made to 250ml with double distilled water.

ProcedurePreparation of extract

1. The spinach leaves were crushed in extraction buffer (20 gm spinach in 80ml buffer).2. The crude extract was filtered through layers of cheese cloth.

Ammonium Salt precipitation1. 100ml of extract was mixed with ammonium sulphate fom initial concenation of 25%to

75%.2. Take 100ml of sample and add14.4 gm ammonium sulphate (25%).Constant stirring is

done to prevent local precipitation.cold temperature is maintained while stiring.3. 15.8 gm ammonium sulphate added to make 50%.4. Finally, the ammonium salt content was made up to 70% .5. Incubate tube overnight at 4 ºC

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6. centrifuge at >2000 rpm, 4 ºC for 20 min.7. Collect the pellets carefully.

Activation of dialysis membrane1. The dialysis membrane was activated by boiling in a solution of 0.9% sodium

bicarbonate and 0.9%of sodium salt of EDTA.2. The boiling was done for 5-10 mins.3. Rinsing was done with solution followed by rinsing in distilled water.4. It was immersed in distilled water for half an hour.5. Store the membrane in 20% ethanol solution.

Dialysis process1. Precipitates obtained after ammonium sulphate precipitation were dissolved in 5ml Tris-

Hcl buffer (pH 7.8).2. Dialysis membrane was immersed in distilled water for 20 minutes.3. Continous washing were given to the membrane with distilled water.4. The membrane was clipped at one side using dialysis clips and checked for any leakage.5. 5ml of the extract was soked in the dialysis membrane using a pipette.6. The membrane was soaked in 200ml of 50mM Tris Hcl buffer pH8.0.7. The dialysis assembly was kept on magnetic stirrer to ensure proper diffusion.8. After one and half hour , the buffer solution was replaced with tris hcl buffer of equal

concentration.9. After overnight incubation, the buffer solution was again replaced.10. On completion of 24 hours of dialysis , the protein sample was removed.11. The sample was stored at 40c.

The same process was carried out after acetone precipitation

a).Regeneration of chromatographic column

1. Sephadex G-25 was dissolved in distilled waer and poured in columm.

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2. It was continuously washed with distilled water for 2-3 times.3. Wash the sephadex column with 20% ethanol. And incubate with ethanol for 1hr.4. Remove the ethanol by washing column with distilled water.5. Wash the column with 0.1 M Nacl solution to remove all ions.6. Maintain the flow of column @ 100 microlitre per minute.

b). Loading the sample 1. 200 microlitre of sample purified after dialysis was dissolved in water and loaded in the

column slowly.2. Ten samples of 500 microlitre each was collected from the columns .

3. Again Bacl2 was carried out for sample obtained after chromatography.4. Estimation of protein activity5. Take 50 microlitre of the samples collected after chromatography.

Estimation of protein activity:

1. Take 50 microlitre of the samples collected after dialysis.2. Make the final volume to 3ml using distilled water.3. Check the optical density at 280nm.

Size Exclusion Chromatography Using Sepharose 6Ba).Regeneration of chromatographic column

1. Sephadex 6B was poured in column (around 5ml of suspension).2. Wash the column with 1.5 M Nacl solution to regenerate the column and remove the

ions.3. Wash with distilled water.4. Maintain the flow of column @ 100 microlitre per minute.

b). Loading the sample 1. 1000 microlitre of sample purified after chromatography using Sephadex G25 was

dissolved in buffer (Tris Hcl pH 8, 50mm) and loaded in the column slowly.2. Samples of 500 microlitre each was collected from the columns .

3. Again Estimation of protein activity4. Take 50 microlitre of the samples collected after chromatography.

Protein estimation at each step of purification using Lowry reagent.1. Test tubes were taken and marked as B,S1, S2,S3,S4,S5,T1,T2,T3,T4,……T8.

2. Add standard BSA (20 mg/100ml) to S1 to S5.

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3. Add 100 microlitre sample to test tubes T 1to T8.

4. Add 4.5 ml of Reagent I and incubate for 10 minutes.5. After incubation add 0.5 ml of reagent II and incubate for 30 minutes6. Measure the absorbance at 660 nm and plot the standard graph .7. Estimate the amount of protein present in the given sample from the standard graph

Barium chloride Test1. 10% Barium chloride solution was prepared and was acidified using a small amount of

Hcl.2. A small volume of protein sample after dialysis , 0.1 ml was added to the salt solution.3. Any resulting changes in solution were observed.

SDS PAGE Protocol

1. Make the separating gel:Set the casting frames (clamp two glass plates in the casting frames) on the casting stands.Prepare the gel solution (as described above) in a separate small beaker.Pipet appropriate amount of separating gel solution (listed above) into the gap between the glass plates.To make the top of the separating gel be horizontal, fill in water.Wait for 20-30min to let it gelate.

2. Make the stacking gel:Discard the water.Pipet in stacking gel untill a overflow.Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate.Make sure a complete gelation of the stacking gel and take out the comb. Take the glass plates out of the casting frame and set them in the cell buffer dam. Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.

3. Prepare the samples:Mix the samples with sample buffer (loading buffer).Add the sample and Tris Hcl buffer pH6.8 and make the final volume of loading buffer to 25 microlitre.Heat them in boiling water for 5-10 min.Load prepared samples into wells and make sure not to overflow.

4. Set an appropriate volt and run the electrophoresis 50-100 volts for 1-1.5 hr.

Observations Barium chloride test

Samples Result

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Protein sample obtained after dialysis of sample obtained after ammonium sulphate precipitation

Positive

Protein sample obtained after dialysis of sample obtained after acetone precipiation

Negative

Samples obtained after Sephadex chromatography1 Positive

2 Positive

3 Negative

4 Negative

5 Negative

6 Negative

7 Negative

8 Negative

9 Negative

10 Negative

Samples obtained after Sepharose chromatography1 Negative

2 Negative

3 Negative

4 Negative

5 Negative

6 Negative

7 Negative

Negative indicates no turbidity visible after addition of barium chloride.

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Protein activity at 280nm

Test sample OD at 280nm

Protein sample obtained after initial extraction

0.44

Protein Sample obtained after ammonium precipitation

0.54

Protein sample obtained after dialysis of sample obtained after ammonium sulphate precipitation

0.700

Samples obtained after chromatography using Sephadex G25

1 0.0

2 0.0

3 0.023

4 0.027

5 0.007

6 0.008

7 0.010

8 0.005

9 0.0

10 0.002

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Samples obtained after chromatography using Sepharose 6B1 0.013

2 0.025

3 .038

4 .093

5 .109

6 .038

7 .038

Protein concentration of sample at 630nm

Sample OD Protein concentration(microgram per microlitre)

S1 0.07S2 0.16S3 0.20S4 0.25S5 0.33T1 sample obtained after initial extraction 0.29 46T2 after ammonium precipitation 0.08 12T3 Protein sample obtained after dialysis of sample obtained after ammonium sulphate precipitation

0.19 30

T4 Protein sample obtained after chromatography using Sephadex G25

0 0

Samples obtained after chromatography using Sepharose 6BT5 0.03 4T6 0.04 6T7 0.03 4T8 0.04 6

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0 10 20 30 40 50 600

0.05

0.1

0.15

0.2

0.25

0.3

0.35

Series2Series4Series6

Protein concentration (microgram/microlitre)

O.D

at 6

60 n

m

References:greproteonics.lifesci.dundec.ac.ukweb.mnstate.edu/provost/AmmoniumSulphateProtocol.pdfweb.mnstate.edu/provost/DialysisProtocol.pdfpiercenet.comProtocol-online.orgdocbrown.infowebformulas.com

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bbc.co.ukgelifesciences.co.jpsolarbio.cnehow.comen.wikipedia.org/wiki/Size-exclusion_chromatographyen.wikipedia.org/wiki/Ammonium_sulfate_precipitationproteomics.ox.ac.ukBidmcmassspec.orgNptel.ac.in/coursesHarvardapparatus.com/guide+for+gel+filterationhttp://www.nizo.com/news/latest-news/28/abundant-plant-protein-extracted-for-food-

application/