3. Protein extraction
Protein
Proteins are the biomolecules,
composed of amino acid, forming the
building block of the system and performs
most of the biological functions of the system.
Protein extraction:
The process by which the proteins from the cell are recovered for
the analysis purpose is called protein extraction
4. Why ?
Proteins are extracted from tissues for a wide range of reasons, including
To compare the structure of proteins as expressed by different
organism.
To purify a protein in order to identify the gene that encodes it, and
To resolve proteins by SDS-PAGE.
To assay an enzyme in a crude extract for physiological studies.
To study the mechanism of action of an enzyme.
To diagnose parasitic diseases.
5. Generic outline for protein isolation
1. Collection of sample, washing, cleaning and grinding to fine powder
2. Cell lysis with physical (Grinding) and chemical (lysis buffers)
methods.
3. Separation of proteins from non-protein components (nucleic acids
and lipids) & recovery of bulk proteins from a crude extract
4. Further separation of target protein-containing fraction from the bulk
proteins
5. Recovery of the target protein in a highly purified state along with a
high yield
6. Materials
Frozen or fresh plant material
Liquid nitrogen
Extraction buffer
Mortar & pestle or tissue grinder
20- to 70-μm pore size nylon mesh
Refrigerated centrifuge and centrifuge tubes
Extraction of Proteins from Plant Tissues
7. procedure
Cleaning and weighing of Leaves
Take isolated leaves in a petri-plate & wash with distilled water to
remove all the particulate matter on the surface.
Dry the leaves on a tissue paper without applying excess pressure.
After the leaves are totally dry, weigh the required amount on a
balance.
8. Liquid Nitrogen Treatment
Take a clean mortar and pestle and place it on ice.
Pre-chill the mortar with liquid nitrogen.
Transfer the leaves into the pre-chilled mortar & Add around 10
ml of liquid nitrogen, all at one go.
9. Grinding the Leaves & Lysis Buffer Treatment
Grind the leaves till a fine powder is seen.
To the powdered leaves, add lysis buffer and grind thoroughly.
Transfer the lysate to fresh eppendorf tubes & Add some more lysis
buffer, to ensure proper lysis of cells.
Vortex the tube thoroughly to mix the contents uniformly.
Protein Precipitation at -20°C
After vortexing the contents, incubate the tube at -20°C for 1
hour.
The proteins at the end of this incubation, get precipitated in the
buffered solution.
10. Centrifugation and Acetone Wash
Take the tube out of -20oC freezer. Centrifuge the tube at 14000
rpm, 4oC for 30 minutes,
Take the tube out of the centrifuge. The tube can be seen to have
a clearly demarcated pellet and supernatant. Discard the
supernatant carefully without disturbing the pellet.
11. To the pellet, add 1ml of wash buffer. Wash buffer helps in
removing the color of the pellet.
Vortex the tube to thoroughly let the pellet dissolve into the buffer.
Incubate the tube in the -20o freezer for 30 minutes.
Remove the tube from the freezer carefully. Centrifuge the tube at
14000rpm, 4oC for 15 minutes.
Discard the supernatant carefully
without disturbing the pellet. Air dry
the pellet to remove even traces of
acetone.
12. Rehydration Buffer Treatment
Add 400 μL of rehydration buffer to the air-dried pellet.
Vortex the tube till the pellet dissolves in the rehydration buffer
(CHAPS solubilizes the proteins, Urea denatures the proteins.)
Incubate the tube overnight at 4oC for complete solubilisation of
proteins in the rehydration buffer.
Sample Storage
The sample contains the protein
If not required immediately it can be stored at -20°C for later use.
13. Protein isolation from animal tissues
Materials
Waring blender
Refrigerated centrifuge
Polypropylene screw-cap
centrifuge tubes
14. Procedure
1. Trim fat and connective tissue from the body and cut into small
pieces
2. Place the tissue in the precooled blender vessel and add cold
extraction buffer(2)
3. Homogenize at full speed for 1—3 min depending on the
toughness of the tissue.
4. Pour the homogenate into a glass beaker, place on ice, and stir
for 15-30 min to ensure full extraction.
15. 5. Remove cell debis and other particulate matter from the
homogenate by centrifugation at 4°C.
6. Pour off the supernatant carefully to avoid disturbing sedimented
material. Any fatty material that has floated to the top of the tube
should be removed by filtering the extract through cheese cloth
7. The pellet may be re-extracted with more buffer to increase the
yield.
8. The filtrate obtained at this stage may require further treatment
to remove insoluble material
9. Thus obtained is protein extract.
16. Protein extraction from fungi
Materials
Malt extract agar (MEA)
Glucose yeast medium (GYM)
Pectin broth
Tris-glycine buffer
Pectinase gel
Gel buffer
Electrode buffer
0.1 Malic acid
17. procedure
This protocol describes the extraction of aqueous intracellular proteins
from the filamentous fungus Metarhizium anisopliae
Harvest the mycelium from given culture by vacuum-assisted filtration onto
Whatman No. 3 filter paper.
Wash the mycelium once in the Buchner funnel with sterile deionized water,
and transfer the harvested mycelium from the filter paper to a plastic Petri dish
with a sterilized spatula.
Freeze the mycelium at -20°C for 24 h.
18. Disrupt freeze dried mycelium by briefly grinding it in a mortar
and pestle & collect it in sterile 1.5 mL micro-centrifuge tubes
(~500-mg). Rehydrate it in 1 mL of Tris-glycine buffer.
Clarify the slurry by centrifugation at 12,500 g for 40 min at 4°C.
After centrifugation, collect the supernatant into another sterile
micro-centrifuge tube
The collected supernatant will contain the total cytoplasmic
proteins. The samples as prepared here typically contain 15-100
mg/mL protein
19. Protein extraction form Bacteria
Materials
Lysis buffer
Hen egg lysozyme
DNase 1
Sodium deoxycholate
MgCl2
20. procedure
1. Harvest the bacterial cells by centrifugation at 1000g for 15
min at 4°C, and pour off the supernatant.
2. Weigh the wet pellet.
3. Add approx 3 mL of lysis buffer for each wet gram of bacterial
cell pellet and resuspend.
4. Add lysozyme to a concentration of 300 µg/mL and stir the
suspension for 30 min at 4°C
21. 5. Add deoxycholate to a concentration of 1 mg/mL while stirring.
6. Place at room temperature, and add DNase 1 to a concentration of
10 mg/mL and MgCl2 to 10 mM. Stir suspension for a further 15
min to remove the viscous nucleic acid
7. Centrifuge the suspension at 10,000g for 15 min at 4°C.
8. Resuspend the pellet in lysis buffer to the same volume as the
supernatant, and analyze aliquots of both for the protein of
interest on SDS-PAGE
22. SDS-PAGE analysis
SDS-PAGE : sodium dodecyl sulfate
polyacrylamide gel electrophoresis
It is a common technique used to separate,
visualize, and therefore compare the
relative amount of individual polypeptide
chains contained in different fractions
It yields the mass of each individual
subunit derived from the denatured
complex
23. procedure
Denaturation:
A small sample from each fraction is first mixed with an excess of
SDS, ßME and bromophenol blue.
Each mixture is then placed in a boiling water bath for several
minutes
In the end, each polypeptide chain is unfolded into a negatively-
charged, rod-shaped complex with a relatively constant charge
to mass ratio.
24. Electrophoresis:
Take a solid, rectangular gel of
polyacrylamide that is cross-
linked to a desired mesh or size
exclusion limit.
A small aliquot from each
denatured sample is transferred
to separate wells of gel.
An electric field is applied across the loaded gel with the positive pole
positioned on the opposite side of the samples
25. Negatively-charged complexes get drived the through the mesh of
polyacrylamide which effectively filters them according to the length of
their Stokes radius as they wiggle through the porous matrix to the
opposite side of the gel.
Small particles move through
the gel more rapidly than larger
ones,
The electrophoresis is visually
followed. Power supply is shut off before any protein in the sample reaches
the bottom of the gel.
26. Staining and destaining:
Following electrophoresis, the entire gel is soaked in a stain
solution containing a dye that tightly binds to the backbone of
each denatured polypeptide chain (usually Coomassie brilliant
blue).
Excess stain is then washed from the gel by soaking it in a destain
solution long enough for the blue-stained bands of each
polypeptide to be visualized against the clear, colorless
background of the gel.
27. PAGE of a sample proteins
stained with Coomassie
blue
Two SDS-PAGE-gels after a completed run
28. After staining, different species
biomolecules appear as distinct bands
within the gel.
It is common to run molecular weight
size markers of known molecular
weight in a separate lane in the gel to
calibrate the gel
Approximate molecular mass of
unknown biomolecules is determined
by comparing the distance traveled
relative to the marker.
Picture of an SDS-PAGE. The
molecular markers (ladder) are
in the left lane
29. protein concentration by spectrophotometer
This method is recommended for pure protein solutions
Principle
Proteins in solution absorb UV light with absorbance maxima at
280 and 200 nm. Amino acids with aromatic rings are the
primary reason for the absorbance peak at 280 nm. Peptide
bonds are primarily responsible for the peak at 200 nm.
30. Procedure
1. Warm up the UV lamp (about 15 min.)
2. Adjust wavelength to 280 nm
3. Calibrate to zero absorbance with
buffer solution only
4. Measure absorbance of the protein solution
5. Adjust wavelength to 260 nm
6. Calibrate to zero absorbance with buffer
solution only
7. Measure absorbance of the protein solution
31. Analysis
Unknown proteins or protein mixtures.
Concentration(mg/ml) =
Absorbance at 280 nm
path length (cm.)
Pure protein of known absorbance coefficient: for 1cm path length
concentration =
Absorbance at 280 nm
absorbance coefficient
32. Concentration may in mg/ml, %, or molarity depending on type
coefficient is used.
Mg protein/ml =
% protein
10
=
molarity
Protein Molecular weight
Unknowns protein with possible nucleic acid contamination.
Conc. (mg/ml) = (1.55 x A280) - 0.76 x A260)