Glutaraldehyde (Pentanedial)

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(General & chemistry)
Current revision (15:39, 8 November 2011) (view source)
(Properties)
 
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* crosslinks are irreversible unlike those of PFA
* crosslinks are irreversible unlike those of PFA
* larger molecule than methanal from PFA & therefore slower penetration into tissue
* larger molecule than methanal from PFA & therefore slower penetration into tissue
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* '''toxic & irritating''' - handle in a chemical fume hood; do not dispose of in the sink
+
* '''toxic & irritating''' - handle in a chemical fume hood; do not dispose of in the sink (collect or inactivate w milk powder)
* causes more autofluorescence than PFA; see [[Griffin:Immunofluorescence_Cell_Staining#Fixative-induced_Fluorescence|Griffin IHC notes]]
* causes more autofluorescence than PFA; see [[Griffin:Immunofluorescence_Cell_Staining#Fixative-induced_Fluorescence|Griffin IHC notes]]
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== Recipes ==
== Recipes ==
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==== 2% GA, 2% PFA in 50mM [[HEPES]] ====
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==== 2% GA, 2% [[PFA]] in 50mM [[HEPES]] ====
<pre>
<pre>
- 6.25  ml    16% aqueous PFA stock  (final 2% v/v)
- 6.25  ml    16% aqueous PFA stock  (final 2% v/v)
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* [[Image:3stars.png]] excellent description of [http://publish.uwo.ca/~jkiernan/formglut.htm PFA & GA fixation by John Kiernan]
* [[Image:3stars.png]] excellent description of [http://publish.uwo.ca/~jkiernan/formglut.htm PFA & GA fixation by John Kiernan]
* [http://en.wikipedia.org/wiki/Glutaraldehyde Glutaraldehyde] and [http://en.wikipedia.org/wiki/Fixation_%28histology%29 Fixation] Wikipedia entries
* [http://en.wikipedia.org/wiki/Glutaraldehyde Glutaraldehyde] and [http://en.wikipedia.org/wiki/Fixation_%28histology%29 Fixation] Wikipedia entries
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* publication on GA crosslinking chemistry: Migneault '04 PMID 15560135, Wine '07 PMID 17461426, Cheung '82 PMID 6299648
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* publications on GA crosslinking chemistry: Migneault '04 PMID 15560135, Wine '07 PMID 17461426, Cheung '82 PMID 6299648
==== Specific applications ====
==== Specific applications ====

Current revision

Chemical structure of glutaraldehyde (1,5-pentane-dial)
Chemical structure of glutaraldehyde (1,5-pentane-dial)

Glutaraldehyde (1,5-pentanedial, abbreviated as GA) is a common fixative in biology. It is used to reduce degradation in cells, tissues, and entire organisms before further experiments like electron microscopy. Fixation occurs by crosslinking (creating covalent chemical bonds between proteins in/on cells). GA is similar to another common cross-linking fixative, PFA.

Contents

Properties

  • glutaraldehyde is a potent, cross-linking fixative
  • bridges larger distances than PFA
  • crosslinks are irreversible unlike those of PFA
  • larger molecule than methanal from PFA & therefore slower penetration into tissue
  • toxic & irritating - handle in a chemical fume hood; do not dispose of in the sink (collect or inactivate w milk powder)
  • causes more autofluorescence than PFA; see Griffin IHC notes

Uses

  • Glutaraldehyde is used to fix specimen before electron microscopy where it is employed alone or mixed with polymethanal (paraformaldehyde) as the first of 2 fixations followed by osmium tetroxide.
  • Glutaraldehyde is used as an amine cross-linker.
  • Glutaraldehyde is used in SDS-PAGE to fix/crosslink proteins and peptides prior to staining. Gels are treated with a 5% solution for ~30 min, after which it must be thoroughly washed to remove the yellow stain brought about by reacting with free Tris. Alternatively, gels can be washed before fixation.

Recipes

2% GA, 2% PFA in 50mM HEPES

- 6.25  ml    16% aqueous PFA stock  (final 2% v/v)
- 4.0   ml    25% aqueous GA stock   (final 2% v/v)
- to 50 ml    with 50 mM HEPES, pH 7.4
  • use fresh ingredients or frozen stocks to preserve reactivity of aldehydes
  • handle with care, protective wear, and under the hood
  • use within days, store at 4°C

Ordering

prices as of 11/2012

  • EM grade (cooled, N2 atmosphere, filtered; so called Grade 1) 25% GA(aq): Sigma 1-2€/ml depending on volume [1]
  • non-EM grade (not refrigerated en route, not purified; Grade 2) 25% GA(aq): Sigma 7-25c/ml depending on volume [2]
  • EM grade (?) from cheaper supplier: Roth 3c/ml [3]

Links

General & chemistry

Specific applications

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