Cell and tissue lysis hub

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
Current revision (12:44, 18 June 2013) (view source)
(synonom addition, typo)
 
(10 intermediate revisions not shown.)
Line 1: Line 1:
-
[[Image:Hub icon.png|right|thumbnail|protocol hub]]
+
[[Image:Hub icon.png|right]]
-
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 ==
Line 9: Line 9:
* 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 ===
Line 22: Line 34:
* 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 ==
-
{| {{table}}
+
{| {{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)
-
| eukaryotic cells
+
| 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)
-
| cell line
+
| cells
|-
|-
| [[Streptomyces:Protocols/Mini-Maxi Prep]]
| [[Streptomyces:Protocols/Mini-Maxi Prep]]
Line 61: Line 74:
| tissue
| tissue
|-
|-
-
| add another method's name
+
| [[Eccles:Protein Lysates from Cells in Culture]]
-
| target
+
| protein
-
| lysis method
+
| detergent (SDS, NP40)
-
| type of starting material
+
| cells
|-
|-
-
| add another method's name
+
| [[Eccles:Protein Lysates from Tissue]]
-
| target
+
| protein
-
| lysis method
+
| detergent, physical, freezing
-
| type of starting material
+
| 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

Current revision

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.

Contents

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

Related hub pages

Related discussions on BioForum

Personal tools