1. 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)
2. 2
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……………………………………………………
3. 3
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 ofBiotecnology, 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 excelevery 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,
4. 4
CERTIFICATE
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)
5. 5
Introduction
RUBISCO
Ribulose-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.
6. 6
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.
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 system
The 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).
Additives
7. 7
Depending 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.
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 solubility
The 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:
8. 8
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.
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 precipitation
Ammonium 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.
PRINCIPLE
Ammonium 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).
9. 9
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.
The commonly used salt is ammonium sulfate, as
1. 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).
ADVANTAGES
1. 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 and
4. 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.
10. 10
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.
PRINCIPLE
Dialysis 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
11. 11
diffuse through a semi-permeable membrane into a second chamber of liquid or dialysate.
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.
12. 12
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 reagent
Alcohol helps to prevent super saturation.
Size-exclusion chromatography
Size-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:
13. 13
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.
14. 14
sephadexG-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
15. 15
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 6B
Sepharose 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 suspension
Sepharose 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 Regeneration
Sepharose 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 strengths
exceeding 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 BYLOWRY’S METHOD
PRINCIPLE:
The principle behind the Lowry method of determining protein
concentrations 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
16. 16
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 PAGE
SDS-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 method
for analysing protein mixtures qualitatively. It is particularly useful for monitoring
protein purification and, because the method is based on the separation of proteins
according to size. Samples to be run on SDS–PAGE are firstly boiled for 5 min in sample buffer
containing
b-mercaptoethanol and SDS. The mercaptoethanol reduces any disulphide bridges
present that are holding together the protein tertiary structure, and the SDS binds
strongly to, and denatures, the protein. Each protein in the mixture is therefore fully
denatured by this treatment and opens up into a rod-shaped structure with a series of
negatively charged SDS molecules along the polypeptide chain. On average, one SDS
molecule binds for every two amino acid residues. The original native charge on the
molecule 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 chain
would result in repulsion between negative charges on different parts of the protein
chain, returning the conformation back to the rod shape.
Once the
samples are all loaded, a current is passed through the gel. The samples to be separated
are not in fact loaded directly into the main separating gel. When the main separating
gel (normally about 5 cm long) has been poured between the glass plates and allowed
17. 17
to set, a shorter (approximately 0.8 cm) stacking gel is poured on top of the separating
gel and it is into this gel that the wells are formed and the proteins loaded. The purpose
of this stacking gel is to concentrate the protein sample into a sharp band before it
enters 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 the
protein the more easily it can pass through the pores of the gel, whereas large proteins
are 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 therefore
indicates the electrophoresis front. When the dye reaches the bottom of the gel, the
current is turned off, and the gel is removed from between the glass plates and shaken
in 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 run
at 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 overnight
destaining is needed to completely remove all background stain. Vertical slab gels
are invariably run, since this allows up to 10 different samples to be loaded onto a
single 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
18. 18
Sephadex G-25
Barium chloride
Reagents
o 2% Na2CO3 in 0.1 N NaOH
o 1% NaK Tartrate in H2O
o 0.5% CuSO4.5 H2O in H2O
o Reagent I: 48 ml of A, 1 ml of B, 1 ml C
o Reagent II- 1 part Folin-Phenol [2 N]: 1 part water
o 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
19. 19
TEMED 2l
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.
Procedure
Preparationof 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 precipitation
1. 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.
20. 20
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
6. centrifuge at >2000 rpm, 4 ºC for 20 min.
7. Collect the pellets carefully.
Activation of dialysis membrane
1. 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 process
1. 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
21. 21
a).Regeneration of chromatographic column
1. Sephadex G-25 was dissolved in distilled waer and poured in columm.
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 activity
5. 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 ExclusionChromatography Using Sepharose 6B
a).Regenerationofchromatographic 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 activity
4. Take 50 microlitre of the samples collected after chromatography.
22. 22
Proteinestimation at eachstep 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.
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 minutes
6. 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 Test
1. 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
23. 23
Barium chloride test
Samples Result
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
chromatography
1 Positive
2 Positive
3 Negative
4 Negative
5 Negative
6 Negative
7 Negative
8 Negative
9 Negative
10 Negative
Samples obtained after Sepharose
chromatography
1 Negative
2 Negative
3 Negative
4 Negative
5 Negative
6 Negative
7 Negative
24. 24
Negative indicates no turbidity visible after addition of barium chloride.
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
25. 25
9 0.0
10 0.002
Samples obtained after chromatography
using Sepharose 6B
1 0.013
2 0.025
3 .038
4 .093
5 .109
6 .038
7 .038
Protein concentrationof sample at 630nm
Sample OD Protein concentration
(microgram per
microlitre)
S1 0.07
S2 0.16
S3 0.20
S4 0.25
S5 0.33
T1 sample obtained after initial extraction 0.29 46
T2 after ammonium precipitation 0.08 12
T3 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 6B
T5 0.03 4
T6 0.04 6
T7 0.03 4
T8 0.04 6
