Glutaraldehyde (Pentanedial): Difference between revisions
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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 | ||
* '''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 == | ||
==== 2% GA, 2% PFA in 50mM [[HEPES]] ==== | ==== 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) | ||
| Line 27: | Line 27: | ||
* handle with care, protective wear, and under the hood | * handle with care, protective wear, and under the hood | ||
* use within days, store at 4°C | * 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 [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=de&N4=G5882|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC] | |||
* non-EM grade (not refrigerated en route, not purified; Grade 2) 25% GA(aq): Sigma 7-25c/ml depending on volume [http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=de&N4=G6257|SIAL&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC] | |||
* EM grade (?) from cheaper supplier: Roth 3c/ml [http://www.carlroth.com/catalogue/catalogue.do;jsessionid=130994B46A65734D5956120A5BB7CCFD?favOid=000000010000187100020023&act=showBookmark&lang=de-de&market=DE] | |||
== Links == | == Links == | ||
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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 | ||
* | * publications on GA crosslinking chemistry: Migneault '04 PMID 15560135, Wine '07 PMID 17461426, Cheung '82 PMID 6299648 | ||
==== Specific applications ==== | ==== Specific applications ==== | ||
Latest revision as of 13:39, 8 November 2011
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.
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
excellent description of PFA & GA fixation by John Kiernan- Glutaraldehyde and Fixation Wikipedia entries
- publications on GA crosslinking chemistry: Migneault '04 PMID 15560135, Wine '07 PMID 17461426, Cheung '82 PMID 6299648
Specific applications
- EM protocol by the Bitan lab using glutaraldehyde as fixative
- Mixed 2% PFA, 0.2% GA for X-gal stainings
- Recipes for a mixed fixative containing 0.1% glutaraldehyde from the O'Neill lab
- Glutaraldehyde as disinfectant
