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. The ups and downs of various methods from sonication, homogenization, freeze-thaw cycles, and detergent-based lysis are examined below. | 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. | ||
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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 | |||
=== 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 == |
Revision as of 03:45, 19 December 2008
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
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 |