Cell and tissue lysis hub: Difference between revisions
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This page should point you to the many different general and lab-specific protocols describing tissue and cell lysis and serve as forum for comparison. | This page should point you to the many different general and lab-specific protocols describing tissue and cell lysis and serve as forum for comparison. The ups and downs of various methods from sonication, homogenization, freeze-thaw cycles, and detergent-based lysis are examined below. | ||
== Comparison of lysis methods == | == Comparison of lysis methods == | ||
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* problem: heat build up which can denature proteins (proportionate to length of sonication) | * problem: heat build up which can denature proteins (proportionate to length of sonication) | ||
* precaution: do on ice and sonicate intermittantly | * precaution: do on ice and sonicate intermittantly | ||
material: ultrasonication bath or rods | |||
=== Homogenisation === | === Homogenisation === | ||
* best for animal tissue; less suitable for cells | * best for animal tissue; less suitable for cells | ||
* precaution: do on ice to reduce heat build-up and denaturation | * precaution: do on ice to reduce heat build-up and denaturation | ||
material: drills (polytron), pestle/tube (dounce) bead beaters | |||
=== Tissue lysis in liquid nitrogen === | |||
* also known as cryo-pulverization | |||
* best for tissue fragments (e.g. biopsy samples, tumor samples) or plant samples | |||
* involves chilling the sample in liquid nitrogen and immediately afterwards subjecting to impact or grinding | |||
* low cost, manual methods require a mortar and pestle or specialized stainless steel devices known as tissue pulverizers | |||
* higher cost, mechanised methods involve a variety of specialized cryogenic cell disruption machines | |||
* plus: good protein extraction from small, tough samples | |||
* negative: using some devices causes residual contamination between samples, not suitable for PCR amplification | |||
=== Freeze-thaw === | === Freeze-thaw === | ||
* least effective method | * least effective method | ||
* plus: does not denature proteins as much as other methods | * plus: does not denature proteins as much as other methods | ||
material: pestle and mortar, liquid nitrogen | |||
=== Detergents === | === Detergents === | ||
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* problems: detergent may inhibit subsequent reactions | * problems: detergent may inhibit subsequent reactions | ||
* problems: detergent may disrupts protein interactions | * problems: detergent may disrupts protein interactions | ||
no additional material required, just the chemicals and typical tubes | |||
== Specific protocols == | == Specific protocols == | ||
{| {{ | {| {{sorttable}} | ||
! style="background:lightgrey"|description/link | ! style="background:lightgrey"|description/link | ||
! style="background:lightgrey"|target | ! style="background:lightgrey"|target | ||
| Line 34: | Line 47: | ||
| protein | | protein | ||
| detergent (Triton) | | detergent (Triton) | ||
| | | cells | ||
|- | |- | ||
| [[Sauer:Lysing E. coli with Lysozymes]] | | [[Sauer:Lysing E. coli with Lysozymes]] | ||
| Line 49: | Line 62: | ||
| protein | | protein | ||
| detergent (RIPA buffer) | | detergent (RIPA buffer) | ||
| | | cells | ||
|- | |- | ||
| [[Streptomyces:Protocols/Mini-Maxi Prep]] | | [[Streptomyces:Protocols/Mini-Maxi Prep]] | ||
| Line 61: | Line 74: | ||
| tissue | | tissue | ||
|- | |- | ||
| | | [[Eccles:Protein Lysates from Cells in Culture]] | ||
| | | protein | ||
| | | detergent (SDS, NP40) | ||
| | | cells | ||
|- | |||
| [[Eccles:Protein Lysates from Tissue]] | |||
| protein | |||
| detergent, physical, freezing | |||
| tissue | |||
|- | |||
| [[RNA extraction using trizol/tri]] | |||
| RNA | |||
| phenol | |||
| cells | |||
|- | |- | ||
| | | [[Sauer:RNA Purification from E. coli]] | ||
| | | RNA | ||
| | | various options | ||
| | | bacteria | ||
|- | |- | ||
| add another method's name | | add another method's name | ||
| Line 75: | Line 98: | ||
| lysis method | | lysis method | ||
| type of starting material | | type of starting material | ||
|} | |||
== Related hub pages == | |||
* [[RNA extraction]] | |||
== Related discussions on BioForum == | == Related discussions on BioForum == | ||
Latest revision as of 09:44, 18 June 2013
This page should point you to the many different general and lab-specific protocols describing tissue and cell lysis and serve as forum for comparison. The ups and downs of various methods from sonication, homogenization, freeze-thaw cycles, and detergent-based lysis are examined below.
Comparison of lysis methods
Sonication
- most efficient method of cell fractionation
- problem: heat build up which can denature proteins (proportionate to length of sonication)
- precaution: do on ice and sonicate intermittantly
material: ultrasonication bath or rods
Homogenisation
- best for animal tissue; less suitable for cells
- precaution: do on ice to reduce heat build-up and denaturation
material: drills (polytron), pestle/tube (dounce) bead beaters
Tissue lysis in liquid nitrogen
- also known as cryo-pulverization
- best for tissue fragments (e.g. biopsy samples, tumor samples) or plant samples
- involves chilling the sample in liquid nitrogen and immediately afterwards subjecting to impact or grinding
- low cost, manual methods require a mortar and pestle or specialized stainless steel devices known as tissue pulverizers
- higher cost, mechanised methods involve a variety of specialized cryogenic cell disruption machines
- plus: good protein extraction from small, tough samples
- negative: using some devices causes residual contamination between samples, not suitable for PCR amplification
Freeze-thaw
- least effective method
- plus: does not denature proteins as much as other methods
material: pestle and mortar, liquid nitrogen
Detergents
- chemical method of lysis
- problems: detergent may inhibit subsequent reactions
- problems: detergent may disrupts protein interactions
no additional material required, just the chemicals and typical tubes
Specific protocols
| description/link | target | lysis method | type of material |
|---|---|---|---|
| Silver: Lysate for Western | protein | detergent (Triton) | cells |
| Sauer:Lysing E. coli with Lysozymes | lysozyme | bacteria | |
| Blackburn:Yeast Colony PCR | DNA | NaOH | yeast |
| Jacobs:Protocol Total Protein Isolation Using RIPA Lysis Buffer | protein | detergent (RIPA buffer) | cells |
| Streptomyces:Protocols/Mini-Maxi Prep | DNA | NaOH | bacteria |
| Western Blot/Tissue Preparation | protein | detergent & physical disruption | tissue |
| Eccles:Protein Lysates from Cells in Culture | protein | detergent (SDS, NP40) | cells |
| Eccles:Protein Lysates from Tissue | protein | detergent, physical, freezing | tissue |
| RNA extraction using trizol/tri | RNA | phenol | cells |
| Sauer:RNA Purification from E. coli | RNA | various options | bacteria |
| add another method's name | target | lysis method | type of starting material |
