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Practical No: 07
Protein extraction from plant and animal
tissue samples

Practical No: 07
Practical: Protein extraction from plant and animal tissue samples
Objectives: To gain experience in extraction of protein from a plant and animal tissue
Introduction:
For the characterization of proteins, the protein has to be extracted from the living tissue
...
Different
methods are used for different methods are used for different tissue depending on the type
of tissue as well as type of protein that is going to be extracted
...
Plant tissues do pose
specific problems, which must be taken into account in extraction of proteins
...
Breaking up fresh tissue can be achieved with grinding mortar and pestle or
adding liquid nitrogen to rapidly freeze the material before blending
...
Proteins whose activities are sensitive to
oxidation require extraction in the presence of reducing agent such as dithiothreitol (DTT)
or mercaptoethanol
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175M Tris-HCl PH 8
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3 M DTT)
100% acetone
0
...
0

Method:
One gram of fresh seeds was grinded to a powder with mortar and pestle
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Homogenate was centrifuged at 5000 rpm for 5 min and supernatant was collected into
a 50ml tube
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It was centrifuged at 5000rpm for 15 min to collect precipitated protein, decanted
the supernatant
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The final pellet was Re-suspended in 1 ml 0
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0 and the absorbance was measured at 280 nm
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Table 01: Absorbance values of plant protein for different wavelengths
Sample
Absorbance(
) Concentration
Directly diluted plant protein at 280nm

0
...
0597

Discussion
A large proportion of the dry weight of the mature seeds of many legumes is protein
...

The first step in sample preparation is isolating the protein from a source
...
However, other sources of protein, such as synthetically derived
protein, can also be run through a Western blot
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Loading buffer, which contains SDS
(sodium dodecyl sulfate is added to the protein suspension
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SDS carries a
negative charge
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Ionic detergents contain head groups with either positive (cationic detergents) or negative
charges

(anionic

detergents)
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Solubilized membrane proteins form mixed micelles
with detergents, which shield the hydrophobic (or transmembrane) domain of the protein
from contact with the aqueous buffer
...
Several factors, such as pH, ionic strength, multivalent ions, and organic solvents,
affect the CMC
...
the aggregation number (N),
b
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the cloud point (CP),
e
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Selection of an appropriate buffer:
Factors should be considered when choosing a buffer:
•

pKa and the effects of temperature

•

interactions with other components (such as enzymes and metal ions)

•

compatibility with different purification techniques

•

ultraviolet (UV) absorption

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permeability through biological membranes

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cost

Tris is a chemical with basic properties, having a pKa of 8
...
It can be used to buffer
solutions from drastic pH changes, keeping them in the pH range of 7
...
0
...

Several strategies exist for eliminating these substances from samples
...
Pierce offers a variety of dialysis and desalting
products for performing such buffer exchanges with small or large sample volumes (see
Related Pierce Products)
...
After centrifugation to pellet the precipitated protein, the supernatant
containing the interfering substance is removed and the protein pellet is redissolved in
buffer compatible with the downstream application
...

Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm
...
Secondary, tertiary, and

quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc
...


Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nm
...

In BIURET COLORIMETRIC ASSAY OF SERUM PROTEIN, Proteins react with copper
ions in alkaline solution to form a violet- colored complex that absorbs light at 550 nm
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Biuret reagent is a solution of
CuSO4 in NaOH
...
Therefore, by measuring the concentration of the complex, using
A550 (Absorbance at 550 nm), you are also measuring the concentration of protein
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The first part consists of
establishing a standard curve by using several known quantities of a pure protein
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Since all proteins react with the biuret agent in the same manner, this
standard curve will give us an E value that can be applied to any protein solution reacted
with biuret
...

Bradford Assay and BCA Assay are other methods of calculating the concentration of the
protein sample
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•
•

http://technical@novusbio
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Animal
Proteomics, Dept
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bidmcmassspec
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pdf (accessed on
15th February 2016)

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http://bitesizebio

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