Immunohistochemical analysis of paraffin-embedded human breast carcinoma using p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 in the presence of control peptide (left) or p21 Waf1/Cip1 Blocking Peptide (right).Learn more about how we get our images
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
NOTE: Do not allow slides to dry at any time during this procedure.
For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; follow with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
posted February 2010
revised March 2016
Protocol Id: 283
This peptide is used to block p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 reactivity in immunohistochemistry protocols.
The quality of the peptide was evaluated by reversed-phase HPLC and by mass spectrometry. The peptide blocks p21 Waf1/Cip1 (12D1) Rabbit mAb #2947 signal in immunohistochemistry.
Use as a blocking reagent to evaluate the specificity of antibody reactivity in immunohistochemistry protocols. For immunohistochemistry, add twice the volume of peptide as volume of antibody used in 100 μl total volume. Incubate for a minimum of 30 minutes prior to adding the entire volume to the slide. Recommended antibody dilutions can be found on the product data sheet.
Supplied in 20 mM potassium phosphate (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 1 mg/ml BSA, 5% glycerol and 1% DMSO. Store at –20°C.
The tumor suppressor protein p21 Waf1/Cip1 acts as an inhibitor of cell cycle progression. It functions in stoichiometric relationships forming heterotrimeric complexes with cyclins and cyclin-dependent kinases. In association with CDK2 complexes, it serves to inhibit kinase activity and block progression through G1/S (1). However, p21 may also enhance assembly and activity in complexes of CDK4 or CDK6 and cyclin D (2). The carboxy-terminal region of p21 is sufficient to bind and inhibit PCNA, a subunit of DNA polymerase, and may coordinate DNA replication with cell cycle progression (3). Upon UV damage or during cell cycle stages when cdc2/cyclin B or CDK2/cyclin A are active, p53 is phosphorylated and upregulates p21 transcription via a p53-responsive element (4). Protein levels of p21 are downregulated through ubiquitination and proteasomal degradation (5).
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