Antigen retrieval: Difference between revisions
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Detection techniques using antibodies often fail to work on PFA- or formalin-fixed, paraffin-embedded sections. Antigen retrieval methods can then, in some cases, enable specific antibody detection. They work by reversing some of the chemical modification of epitopes during fixation. These procedures will not help much with epitope loss due to denaturation during sample treatment, like hot paraffin embedding. | Detection techniques using antibodies often fail to work on PFA- or formalin-fixed, paraffin-embedded sections. Antigen retrieval methods can then, in some cases, enable specific antibody detection. They work by reversing some of the chemical modification of epitopes during fixation. These procedures will not help much with epitope loss due to denaturation during sample treatment, like hot paraffin embedding. | ||
== | == Types of retrieval methods == | ||
=== Heat treatment === | |||
* Mechanism: heat cleaves cross-links, exposes buried epitopes of proteins, protein unfolding/refolding | |||
* Specific protocol 1: citrate buffer [http://openwetware.org/wiki/Livesey:_Antigen_retrival][http://www.ihcworld.com/_protocols/epitope_retrieval/citrate_buffer.htm] | |||
* Specific protocol 2: Tris-EDTA buffer [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm] | |||
=== Protease treatment === | |||
* Specific protocol 1: proteinase K [http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm] | |||
* Specific protocol 2: trypsin [http://www.ihcworld.com/_protocols/epitope_retrieval/trypsin.htm] | |||
=== Detergent treatment === | |||
* Specific protocol: SDS [http://openwetware.org/wiki/Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells] | |||
== Personal and lab-specific protocols == | |||
* Korey Griffin's notes: | |||
:* [[Griffin:Antigen Retrieval Technique]] - overview and comparison of techniques | |||
:* [[Griffin:Immunohistochemistry_Paraffin#Antigen_Retrieval]] - heat-based retrieval | |||
:* [[Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells|Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval]] - detergent-based retrieval | |||
* [[Livesey: Antigen retrival|Livesey's antigen retrival]] - heat-based retrieval | |||
* [[Immunohistochemistry for AntiOsteocalcin Antibody]] - a protocol with a protease-based antigen retrieval | |||
== See also== | |||
* [[Antibodies]] | |||
* [[Griffin:Antibody Basics]] | |||
== External links == | |||
* [[Image:3stars.png]] [http://www.ihcworld.com/_technical_tips/antigen_retrieval_tips.htm detailed description of antigen retrieval methods at IHCWorld] | |||
* [[Image:3stars.png]] [http://www.ncbi.nlm.nih.gov/pubmed/17197287 Yamashita 2007] - 60 page detailed review article including antibody generation, fixation, and details on several antigen retrieval techniques | |||
* [http://www.nordiqc.org/Techniques/Epitope_retrieval.htm review of retrieval methods at NordiQC and examples of which antibodies work with which method] | |||
* [http://www.bioreagents.com/pages/protocols.cfm#3|Antigen retrieval protocols using heat at Thermo] | |||
* [http://www.rockland-inc.com/commerce/misc/Antigen_retrieval_methods.jsp|Heat and protease antigen retrieval at Rockland, Inc.] | |||
# | Commercial antigen retrieval kits: | ||
* [http://www.bdbiosciences.ca/ptProduct.jsp?prodId=19647&catyId=76670 BD Pharmigen Retrievagen A pH 6] and [http://www.bdbiosciences.ca/ptProduct.jsp?prodId=19653&catyId=76670 BD Pharmigen Retrievagen B pH 9.5] | |||
* [http://www.genetex.com/WebPage/Product/ProductDetail.aspx?CatalogItemID=33939 GeneTex antigen retrieval solution (heat-based)] and [http://www.genetex.com/WebPage/Product/ProductDetail.aspx?CatalogItemID=66695 GeneTex antigen retrieval solution (protease-based)] | |||
* [http://www.abcam.com/10x-Heat-Mediated-Antigen-Retrieval-Solution-pH-6-0-ab973.html abcam heat-based antigen retrieval solution 10x] | |||
* [http://www.biocompare.com/ProductListings/17837/Antigen-Retrieval-Solutions.html antigen retrieval search at biocompare] | |||
[[Category:Protocol]] | |||
[[Category:Antibody]] | |||
[[Category:Protein]] | |||
[[Category:In vitro]] | |||
Latest revision as of 12:13, 2 November 2009
| back to protocols | ||
Detection techniques using antibodies often fail to work on PFA- or formalin-fixed, paraffin-embedded sections. Antigen retrieval methods can then, in some cases, enable specific antibody detection. They work by reversing some of the chemical modification of epitopes during fixation. These procedures will not help much with epitope loss due to denaturation during sample treatment, like hot paraffin embedding.
Types of retrieval methods
Heat treatment
- Mechanism: heat cleaves cross-links, exposes buried epitopes of proteins, protein unfolding/refolding
- Specific protocol 1: citrate buffer [1][2]
- Specific protocol 2: Tris-EDTA buffer [3]
Protease treatment
Detergent treatment
- Specific protocol: SDS [6]
Personal and lab-specific protocols
- Korey Griffin's notes:
- Griffin:Antigen Retrieval Technique - overview and comparison of techniques
- Griffin:Immunohistochemistry_Paraffin#Antigen_Retrieval - heat-based retrieval
- Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval - detergent-based retrieval
- Livesey's antigen retrival - heat-based retrieval
- Immunohistochemistry for AntiOsteocalcin Antibody - a protocol with a protease-based antigen retrieval
See also
External links
detailed description of antigen retrieval methods at IHCWorld- Yamashita 2007 - 60 page detailed review article including antibody generation, fixation, and details on several antigen retrieval techniques
- review of retrieval methods at NordiQC and examples of which antibodies work with which method
- retrieval protocols using heat at Thermo
- and protease antigen retrieval at Rockland, Inc.
Commercial antigen retrieval kits:
