Antigen retrieval

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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.
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== Protocol ==
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== Types of retrieval methods ==
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=== Heat treatment ===
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* Mechanism: heat cleaves cross-links, exposes buried epitopes of proteins, protein unfolding/refolding
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* Specific protocol 1: citrate buffer [http://openwetware.org/wiki/Livesey:_Antigen_retrival][http://www.ihcworld.com/_protocols/epitope_retrieval/citrate_buffer.htm]
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* Specific protocol 2: Tris-EDTA buffer [http://www.ihcworld.com/_protocols/epitope_retrieval/tris_edta.htm]
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=== Protease treatment ===
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* Specific protocol 1: proteinase K [http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm]
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* Specific protocol 2: trypsin [http://www.ihcworld.com/_protocols/epitope_retrieval/trypsin.htm]
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=== Detergent treatment ===
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* Specific protocol: SDS [http://openwetware.org/wiki/Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells]
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== Personal and lab-specific protocols ==
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* Korey Griffin's notes:
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:* [[Griffin:Antigen Retrieval Technique]] - overview and comparison of techniques
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:* [[Griffin:Immunohistochemistry_Paraffin#Antigen_Retrieval]] - heat-based retrieval
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:* [[Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells|Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval]] - detergent-based retrieval
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* [[Livesey: Antigen retrival|Livesey's antigen retrival]] - heat-based retrieval
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* [[Immunohistochemistry for AntiOsteocalcin Antibody]] - a protocol with a protease-based antigen retrieval
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== See also==
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* [[Antibodies]]
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* [[Griffin:Antibody Basics]]
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# Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
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== External links ==
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# Leave the chosen slides at room temperature just until the frost melts.
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* [[Image:3stars.png]] [http://www.ihcworld.com/_technical_tips/antigen_retrieval_tips.htm detailed description of antigen retrieval methods at IHCWorld]
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# Hydrate slides in 1x PBS for 10 seconds.
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* [[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
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# Put slides into 0.01 M Citric acid (pH 6.0), microwave just to a boil, and let cool for 5 minutes.
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* [http://www.nordiqc.org/Techniques/Epitope_retrieval.htm review of retrieval methods at NordiQC and examples of which antibodies work with which method]
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# Repeat Step 4 two more times (three times total).
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* [http://www.bioreagents.com/pages/protocols.cfm#3|Antigen retrieval protocols using heat at Thermo]
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# Rinse the slides 6 times in 1x TBST for 8 minutes each.  
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* [http://www.rockland-inc.com/commerce/misc/Antigen_retrieval_methods.jsp|Heat and protease antigen retrieval at Rockland, Inc.]
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# Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
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Commercial antigen retrieval kits:
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# Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
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* [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]
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# Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
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* [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)]
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# Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
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* [http://www.abcam.com/10x-Heat-Mediated-Antigen-Retrieval-Solution-pH-6-0-ab973.html abcam heat-based antigen retrieval solution 10x]
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# Rinse 3 times in 1x TBS for 8 minutes each.
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* [http://www.biocompare.com/ProductListings/17837/Antigen-Retrieval-Solutions.html antigen retrieval search at biocompare]
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# Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
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# Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
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TBST = TBS + 0.1% Triton
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[[Category:Protocol]]
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[[Category:Antibody]]
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[[Category:Protein]]
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[[Category:In vitro]]

Current revision

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.

Contents

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

  • Specific protocol 1: proteinase K [4]
  • Specific protocol 2: trypsin [5]

Detergent treatment

  • Specific protocol: SDS [6]

Personal and lab-specific protocols

  • Korey Griffin's notes:

See also

External links

Commercial antigen retrieval kits:

Personal tools