Practical No : 06

Practical : Protein extraction from plant samples

Objectives : To gain experience in extraction of proteins from a plant tissue

Introduction : For the characterization of protein has to be extracted from the living tissue.
Proteins from frozen tissue samples need to be extracted efficiently and without degradation to make the
best use of a limited resource and to ensure, as much as possible, that an accurate representation of the
proteins in the living tissue is obtained. Different methods are used for different tissues depending on the
type of tissue as well as type of protein that is to be extracted.

Materials:

Mortal and pestal

Legume seeds (lantil seeds)

UV/Visible spectrophotometer

SDS extraction buffer (0.175M Tris HCl pH 8.8, 5% SDS, 15% Glycerol, 0.3M DTT)

100% acetone

0.05 M Tris-HCl Buffer, pH 8.0

Method:

1g of fresh Chick-pea seeds was grinded to a powder with mortal and pestle. Then 5ml of SDS
extraction buffer was directly added to the mortal and grinding was continued for as additional
30sec.The homogenate was centrifuged for 5 min at 5000rpm and the supernatant was collected into a
50ml tube.4 volumes of Ice cold 100% acetone were immediately added to recovered supernatant, mixed
by overtaxing and placed at -20’C for one hour to precipitate protein. Then this was centrifuged at
5000rpm for 15min to collect precipitated protein, the supernatant was decant. The final protein from
centrifugation at 5000rpm for 15min was collected and dried the pellet. The pellet was re-suspended in
1ml pH 8.0, 0.05M Tris-HCl buffer and measured the absorbance at 280nm.
Observations:

Table 01: Absorbance values of different protein samples at 280 nm and 550 nm

Type of seed sample Absorbance

At 280 nm At 550 nm

Cow pea 0.823 0.171

Chick pea 0.336 0.121
Dhal 0.215 0.118
Green grams 0.660 0.185
Kidney beans 0.355 0.149
Green peas 0.115 0.177
Urad beans 0.108 0.054
Monkey beans 0.657 0.148

Absorbance of the protein sample extracted from chick pea was read spectrophotometrically.

Absorbance measurement of the chick pea protein sample at 280nm = 0.336

Calculation:

Determination of protein concentration using A280

For an unknown protein or protein mixture, the following formula can be used to obtain a rough estimate
of protein concentration. Using this procedure, protein concentration of a sample from 20µg/ml to 3
mg/ml can be measured.

At 280 nm, 1 Absorbance unit = 1 mg/ ml

Therefore, concentration of the lentil sample when Absorbance is 0.336 = 0.336 x 1 mg/ml

=0.336 mg/ml

The concentration of protein extracted from 1g of chick pea =0.336 mg/ml

Conclusion:

The concentration of the protein sample which is extracted from 1g of chick pea =0.336 mg/ml

Discussion:

Preparation of extracts from plant

Plant cells possess a rigid cell wall and require a great deal of strategy for extraction. Besides cytoplasm,
plant cells contain four organelles: nucleus, plastid, mitochondrion, and vacuole. Vacuole, which
occupies 90% of the cell volume, serves the functions of both a vessel and a lysosome. Vacuoles contain
alkaloids and various hydrolases including proteases. Therefore, protease inhibitors should be used
during preparation of plant extract. The chloroplast is the second major organelle in the green tissues.
Procedures are available for the isolation of chloroplasts, mitochondria, and nuclei from plant cells.

In order to isolate membrane proteins, the first step is to remove contaminating cytosolic proteins.
Soluble cytoplasmic proteins are extracted by cell disruption in a neutral, isotonic, and detergent-free
buffer and are removed after centrifugation. Membrane proteins remain associated with the insoluble
components of the cell extract. In the second step of isolating membrane proteins, a highly purified
membrane fraction is obtained. The starting material is further enriched, if the target protein is known to
be associated with a specific subcellular membrane fraction such as plasma membrane, mitochondria, or
nucleus.

The extraction step is the starting point for all subsequent procedures. In order to isolate intracellular
proteins, cells must be disrupted. Several disruption techniques, both mechanical and chemical, are
available. An efficient method for cell disruption must be developed to release the protein in a soluble
form from its intracellular compartment. The disruption protocol should be as gentle as possible to the
protein. The success of cell disruption depends on a number of variables, such as the choice of buffers,
the presence of protease inhibitors, and the osmolarity of the resuspension buffer. The condition and the
constituent of the extraction buffer depend on the nature of the cell type, the target protein, and its
intended application.

Mechanical lysis for protein extraction

Lysis can be done either by mechanically or chemically. In this practical we used mechanical lysis
process. Mechanical lysis means disruption of cells using sonication, a pressure cell, homogenizer, or
bead beater. Mechanical lysis methods are economical and preferable for large-scale preparations
because the addition of chemicals is not required. However, mechanical lysis produces heat, which
needs to be controlled. Care should also be taken to avoid foaming, to prevent surface denaturation and
oxidation. In our practical Cell lysis was performed by hand, using mortar and pestle.

Use of detergents in extraction

Detergents are generally added to the buffer for extraction and purification of membrane proteins, which
are usually insoluble in an aqueous buffer. Detergents are a class of compounds characterized by their
amphiphilic structure (both hydrophobic and hydrophilic). The “tail” of the detergent molecule is
hydrophobic, usually consisting of a linear or branched hydrocarbon, whereas the hydrophilic head may
have diverse chemical structures. The solution of proteins to be analyzed is mixed with SDS, an anionic
detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative
charge to each protein in proportion to its mass. This allows keeping proteins soluble in solution without
precipitating.

Sodium dodecylsulfate (SDS) extraction buffer : H3 C—(CH 2)11—OSO3 – Na+

Detergents are of three types: ionic, non-ionic, and zwitterionic . Ionic detergents contain head groups
with either positive charges (cationic detergents) or negative charges (anionic detergents). Ionic
detergents denature proteins; thus, these are not included in the extraction. Sodium dodecyl sulfate
(SDS) and cetyltrimethylammonium bromide (CTAB) are examples of anionic and cationic detergents,
respectively.

Removal of detergents from membrane protein

The most membrane proteins precipitate upon complete removal of detergent. However, during the
course of the purification of a membrane protein, it may be necessary to remove excess detergent or
exchange detergent prior to separation by certain charge-based related techniques such as ion-exchange
chromatography and isoelectric focusing. As the initial extraction is carried out with a high amount of
detergent in order to give maximal dispersion of membrane proteins and lipids, removal of excess
detergent at this stage usually facilitates the subsequent purification procedure. The detergent exchange
is best achieved by absorbing of the detergent-membrane protein complex to a chromatographic support
followed by extensive washing with a buffer containing the new detergent.

Reagent/Matrix Principle Remarks

Acetone Protein Precipitation Detergent (e.g., SDS) remains in solution.

After centrifuge at 500rpm for 15 min, SDS detergent remains in solution (supernatant) and Protein has
been precipitated.

Use of Buffer solution

Proteins are extremely heterogeneous biological macromolecules. Their properties can be severely
affected by small changes in hydrogen ion concentration, and thus a stable pH of the protein
environment is necessary.

In order to ensure reproducible experimental results, it is important to maintain the protein solution at
the constant pH. It has been observed that partially neutralized solutions of weak acid or weak bases are
resistant to pH changes on the addition of small amounts of strong acid or strong base. This is known as
“buffering.” Buffer solutions consist of a weak acid and its salt with a strong base, or of a weak base
and its salt with a strong acid. The buffering capacity may be explained by the following equations:

Equation 1: HA (acid) ↔ H+ + A- (incomplete)

Equation 2: NaA ↔ Na + + A- (complete)

NaA is shown here as an example of salt of weak acid and strong base. A is called conjugate base of the
acid HA. The addition of small amounts of strong acid (H +) to the buffer shifts equilibrium (Equation 1)
to the left using A- supplied by Equation 2. Whereas the addition of small amounts of strong base (OH- )
combines with H+ provided by equilibrium (Equation 1) moving to the right. In either case, change of
H+ concentration, hence pH, is unchanged. The pH of a solution of a weak acid and or base is calculated
as follows: From Equation 1, the dissociation constant (K ) is defined as a
This is the Henderso -Hasselbalch equation.

A 20% increase in sensitivity of the assay is achieved when the Folin reagent is added in two portions,
vortexing between additions. The addition of the dithiothreitol 3 min after the addition of the Folin
reagent enhances sensitivity by 50%. Bio-Rad DC (detergent compatible) protein assay is based on the
Lowry assay and can be used to assay protein in the presence of 1% detergent. Bio-Rad RC DC protein
assay, also based on the Lowry assay, is compatible with reducing agent as well as detergent. But, the
sensitivity of both assays is compromised (0.2 to 1.5 mg/ml) compared to the Lowry assay (0.01 to 1
mg/ml). The Lowry assay can be performed in a few seconds by using microwave irradiation. After
irradiation, the absorbance of each sample and standard is read spectrophotometrically.

References

1. Baydoun, AR., ‘Cell Culture Techniques’, in Keith Wilson & John Walker (ed.) 1997, Principles
and techniques of biochemistry and molecular biology,7th edn, Cambridge University Press, pp.
38-72.
2. Walker,J, ‘Protein structure, purification, characterisation and function analysis’, in Keith
Wilson & John Walker (ed.) 1997, Principles and techniques of biochemistry and molecular
biology,7th edn, Cambridge University Press, pp. 300-51.
3. Amersham Pharmacia Biotech AB 1999, Protein purification handbook, AB edn.
4. Alastair Aitken & Michele Learmonth , ‘Protein Determination by UV Absorption’ ,in John M.
Walker (ed) 1996, The protein protocols handbook, Humana Press, New Jersey, pp.3-7.