Download A Review on Cell Lysis, Fractionation and Cellular Content Extraction

A REVIEW ON
CELL LYSIS, FRACTIONATION AND
CELLULAR CONTENT EXTRACTION
PURIA RAFSANJANI NEJAD, PARSA NAMAKI ARAGHI, MEHDI POURNIA
GUIDED BY DR. H. SADRABADI
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LYSIS
• Every cell has a plasma membrane, a protein-lipid bilayer that acts like a barrier, separating
cellular contents from the extracellular matrix. Lipids comprising the plasma membrane are
amphipathic, having hydrophilic and hydrophobic parts that combine spontaneously to form a
closed bimolecular sheet.
• Lysis ("to unbind") refers to the breaking down of a cell, often by viral, enzymic, or osmotic
mechanisms that compromise its integrity.
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MECHANICAL METHODS
Sonication
• Cells are lysed by liquid shear and cavitation.
• The main problem is controlling the temperature. This is
addressed by keeping the suspension on ice and using a
number of short pulses (5-10 sec) with pauses (10-30 sec)
to re-establish a low temperature.
• For cell quantities larger than 50g the method is of
limited value.
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MECHANICAL METHODS
Homogenization
• Homogenizers are the most common devices to lyse bacteria
• Cell lysis occurs by pressurizing the cell suspension and suddenly
releasing the pressure.
• 6000-10,000 psi.
• Multiple (2-3) passes are generally required to achieve adequate
lysis.
• results in a rise in operating temperatures.
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MECHANICAL METHODS
Freezing and grinding:
• The cells are frozen directly in liquid nitrogen
and then are ground to a powder
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MECHANICAL METHODS
Freeze/Thaw:
• Freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material
at room temperature or 37°C.
• Causes cells to swell and ultimately break as ice crystals form during the freezing process and
then contract during thawing.
• Multiple cycles are necessary for efficient lysis
• The process can be quite lengthy
• Has been shown to effectively release recombinant proteins located in the cytoplasm
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CHEMICAL METHODS:
Detergent cell lysis is a milder and easier alternative to physical disruption of cell membranes,
although it is often used in conjunction with homogenization and mechanical grinding.
lipid-lipid, protein-protein and protein-lipid interactions are disrupted.
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CHEMICAL METHODS:
RIPA: RIPA buffer is a popular choice for lysis and
protein extraction of mammalian cells or soft tissue.
RIPA buffer is effective when the immediate
downstream application is SDS-PAGE
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CHEMICAL METHODS:
IP-compatible Solutions:
When the immediate downstream application for a
lysate is a protein affinity purification it is to ensure that
the lysis reagent does not contain denaturants or
components that might interfere with the antibody
binding or protein interactions of interest
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VIRTUAL DNA EXTRACTION
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CELL LYSIS IN MICROFLUIDICS
Mechanical lysis employs cellular contact forces
to crush or burst the cells.
(A) Single cell immobilized in a random array of micropillar obstacles, (B) detailed fluorescent
image of DNA strands looped around the micropillars and suspended by hydrodynamic flow, and
(C) stretched DNA strands from the cell shown in (A). The multiply folded strands extend over
more than 10 mm into the microchannel.
Microfluidic extraction, stretching and analysis of human chromosomal DNA from single cells,
Jaime J. Benítez el al, Lab Chip. Nov 21, 2012; 12(22): 4848–4854.
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OUR IDEA
In 2
In 1
Out 1
Out 2
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CELL LYSIS IN MICROFLUIDICS
Thermal lysis uses high temperatures to disrupt the cell membrane.
Thermal Lysis is one of the more well-established lysis techniques in nucleic acid preparation.
The short exposure is sufficient to cause significant damage to the cell membrane without
damaging the nucleic acids.
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CELL LYSIS IN MICROFLUIDICS
Chemical lysis uses a chemical buffer or enzymes to break down the cell membrane.
The basis is the same with bulk chemical method.
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CELL LYSIS IN MICROFLUIDICS
Electrical lysis induces cell membrane porosity with a low-strength electric field or complete
lysis of the cells with a stronger field.
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CELL LYSIS IN MICROFLUIDICS
The goal for the microfluidics community has been to generate fully integrated systems that can
complete an assay from sample-in to answer-out.
There have been surprisingly few ‘‘complete’’ lab-on-a-chip systems in recent years that can deal
with raw samples, and that is primarily because of the lack of development in sample preparation
systems.
The primary challenge with many of these systems is complexity.
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REFERENCES
• 1)jungkyu kim,wa michael johnson,wb parker hilla and bruce K. Gale*b, integrative biology,
2009, microfluidic sample preparation: cell lysis and nucleic acid purification
• 2)cell lysis technical handbook, thermo scientific, http://www.Piercenet.Com/page/cell-lysistechnical-handbook-1601757
• Jaime J. Benítez et al, Microfluidic extraction, stretching and analysis of human chromosomal
DNA from single cells, Lab Chip, Mar 14, 2014.
• 3)http://www.Embl.De/pepcore/pepcore_services/protein_purification/extraction_clarification/cel
l_lysates_ecoli/
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Thank you for your attention !!!!
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