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Phospho-NF-κB p105 (Ser933) (178F3) Rabbit mAb (IHC Specific) #4808
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-NF-kappaB p105 (Ser933) (178F3) Rabbit mAb (IHC Specific) in the presence of control peptide (left) or Phospho-NF-kappaB p105 (Ser933) Blocking Peptide #1021 (right).Learn more about how we get our images
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic localization, using Phospho-NF-kappaB p105 (Ser933) (178F3) Rabbit mAb (IHC Specific).Learn more about how we get our images
Immunohistochemical analysis of paraffin-embedded human colon carcinoma untreated (left) or lambda-phosphatase-treated (right), using Phospho-NF-kappaB p105 (Ser933) (178F3) Rabbit mAb (IHC Specific).Learn more about how we get our images
Gallery: Phospho-NF-κB p105 (Ser933) (178F3) Rabbit mAb (IHC Specific) #4808
A. Solutions and Reagents
- Ethanol, anhydrous denatured, histological grade (100% and 95%)
- Deionized water (dH2O)
- Hematoxylin (optional)
- Wash Buffer:
- 1X Tris Buffered Saline with Tween® 20 (TBST): To prepare 1L 1X TBST add 100 ml 10X Tris Buffered Saline with Tween® 20 (#9997) to 900 ml dH20, mix.
- Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L, add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7â¢2H2O) to 1 L dH2O. Adjust pH to 6.0.
- 3% Hydrogen Peroxide: To prepare, add 10 ml 30% H2O2 to 90 ml dH2O.
- Blocking Solution: TBST/5% normal goat serum (#5425): to 5 ml 1X TBST add 250 µl normal goat serum.
- Detection System: VECTASTAIN® Elite ABC, including biotinylated secondary antibody (Vector Laboratories).
- Substrate: Vector® NovaRED™ (Vector Laboratories).
NOTE: Do not allow slides to dry at any time during this procedure.
- Deparaffinize/hydrate sections:
- Incubate sections in three washes of xylene for 5 minutes each.
- Incubate sections in two washes of 100% ethanol for 10 minutes each.
- Incubate sections in two washes of 95% ethanol for 10 minutes each.
- Wash sections twice in dH2O for 5 minutes each.
C. Antigen Unmasking
For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer, pH 6.0; maintain at a sub-boiling temperature for 10 min. Cool slides on bench top for 30 min.
- Wash sections in dH2O three times for 5 minutes each.
- Incubate sections in 3% hydrogen peroxide for 10 minutes.
- Wash sections in dH2O twice for 5 minutes each.
- Wash sections in wash buffer for 5 minutes.
- Block each section with 100-400 µl blocking solution for 1 hour at room temperature.
- Remove blocking solution and add 100-400 µl primary antibody diluted in blocking solution to each section. Incubate overnight at 4°C.
- Prepare ABC solution per manufacturer's recommendations.
- Remove primary antibody and wash section three times with wash buffer for 5 minutes each.
- Add 100-400 µl biotinylated secondary antibody, diluted in TBST per manufacturer’s recommendation, to each section. Incubate 30 minutes at room temperature.
- Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
- Cover sections with 100-400 µl pre-mixed ABC solution as needed and incubate in a humidified chamber for 30 min at room temperature.
- Wash section three times with wash buffer for 5 min each.
- Prepare Vector® NovaRED™ per manufacturer's recommendations.
- Apply 100-400 µl substrate to each section and monitor closely. 5-15 minutes generally provides an acceptable staining intensity.
- If desired, counterstain sections in hematoxylin per manufacturer’s instructions.
- Wash sections in dH2O two times for 5 minutes each.
- Dehydrate sections:
- Incubate sections in 95% ethanol two times for 10 seconds each.
- Repeat in 100% ethanol, incubating sections two times for 10 seconds each.
- Repeat in xylene, incubating sections two times for 10 seconds each.
- Mount coverslips.
posted June 2005
revised February 2008
protocol id: 310
Phospho-NF-kappaB p105 (Ser933) (178F3) Rabbit mAb detects endogenous levels of p105NF-kappaB only when phosphorylated at serine 933.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with a synthetic phosphopeptide corresponding to amino acids around Ser933 of NF-kappaB p105.
Transcription factors of the nuclear factor κB (NF-κB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, c-Rel, RelB, NF-κB1 (p105/p50), and NF-κB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).
Following IKK-mediated phosphorylation of p105 NF-kappaB at multiple sites (Ser921, 923, 927 and 933) on its carboxy-terminus, SCFbeta-TrCP mediated processing produces the 50 kDa active form p50 (12,13).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc. U.S. Patent No. 5,675,063.