Antigen retrieval

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(external links)
Current revision (14:13, 2 November 2009) (view source)
(External links)
 
(5 intermediate revisions not shown.)
Line 4: Line 4:
== Types of retrieval methods ==
== Types of retrieval methods ==
-
# Heat treatment
+
=== Heat treatment ===
-
# Protease treatment
+
* Mechanism: heat cleaves cross-links, exposes buried epitopes of proteins, protein unfolding/refolding
-
# Detergent treatment
+
* 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]
-
== Sample heat-treatment protocol using a citrate buffer ==
+
=== Protease treatment ===
-
# Choose desired slides (paraformaldyhyde-fixed, 10 micron thick crysections).
+
* Specific protocol 1: proteinase K [http://www.ihcworld.com/_protocols/epitope_retrieval/proteinase-k.htm]
-
# Leave the chosen slides at room temperature just until the frost melts.
+
* Specific protocol 2: trypsin [http://www.ihcworld.com/_protocols/epitope_retrieval/trypsin.htm]
-
# Hydrate slides in 1x PBS for 10 seconds.
+
 
-
# Put slides into '''0.01 M Citric acid (pH 6.0), microwave''' just to a boil, and let cool for 5 minutes.
+
=== Detergent treatment ===
-
# Repeat Step 4 two more times (three times total).
+
* Specific protocol: SDS [http://openwetware.org/wiki/Griffin:Immunofluorescence_Cell_Staining#Antigen_retrieval_for_cryostat_tissue_sections_or_cultured_cells]
-
# Rinse the slides 6 times in 1x TBST for 8 minutes each.
+
-
# Block with 5% milk powder in 1x TBST for 30 minutes at room temperature. Optional: 1-2% BSA can be added to TBST.
+
-
# Incubate overnight at 4oC with primary antibodies in TBST plus 5% milk.
+
-
# Rinse for 10 seconds, 1x TBST. Then, rinse 3 times in 1x TBST for 8 minutes each.
+
-
# Add secondary antibodies in TBST for 1 hour at room temperature in the dark.
+
-
# Rinse 3 times in 1x TBS for 8 minutes each.
+
-
# Add Fluorescent Mounting Medium, coverslip, and examine under a fluorescent microscope.
+
-
# Lastly, seal the edges of covered-slip slides with a clear nail polish. This helps slides retain fluorescence longer during storage.
+
-
TBST = TBS + 0.1% Triton
+
== Personal and lab-specific protocols ==
== Personal and lab-specific protocols ==
Line 37: Line 29:
== External links ==
== 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.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.]
* [http://www.rockland-inc.com/commerce/misc/Antigen_retrieval_methods.jsp|Heat and protease antigen retrieval at Rockland, Inc.]

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