Description
Cyclins and cyclin-dependent kinases (cdks) are evolutionarily conserved proteins that are essential for cell-cycle control in eukaryotes. Cyclins (regulatory subunits) bind to cdks (catalytic subunits) to form complexes that regulate the progression of the cell cycle. The activity of these complexes is modulated by activating and inhibitory phosphorylation events, as well as by interactions with small regulatory proteins including, p16, p21, p27, and others. These proteins, referred to as inhibitors of Cdk activity (CdkIs), bind to cyclins, cdks or their complexes. p21, also known as senescent cell-derived inhibitor 1 (Sdi1), wild-type p53-activated fragment 1 (Waf1), Cdk-interacting protein 1 (Cip1), and p53-regulated inhibitor of Cdks (Pic1) inhibits cyclin D-cdk4, cyclin E-cdk3, cyclin A-cdk2, and cyclin A-cdk1. p21 can also inhibit cell cycle progression by binding to PCNA and blocking DNA replication. p21 has also been shown to be a component of active cyclin-cdk complexes, suggesting that p21-containing complexes may shift between active and inactive states through changes in p21 content. Active, p21-containing complexes appear to contain one p21 molecule, whereas inactive compexes contain multiple p21 molecules. The expression of p21 can be induced in response to number of signals, including transcriptional upregulation by the tumor suppressor protein, p53. Human p21 has a calculated molecular weight of 18 kDa and runs at 21 kDa in SDS-PAGE. Clone SXM30 reacts with human, rat, and mouse p21. With regard to cross-species recognition, SXM30 has similar properties to a related p21 clone, SX118. Purified, recombinant human p21-fusion protein was used as immunogen.
Clone SXM30 has been shown to be useful for immunoprecipitation (1-2 µg/1x10^6 cells), western blot analysis (1-2 µg/ml), indirect immunofluorescence microscopy of tissuecultured cells, immunohistochemistry of frozen, acetone-fixed tissue sections (1-5 µg/ml), and paraffin-embedded tissue sections requiring antigen retrieval (20 µg/ml). These applications are similar to that described for a related p21 clone, SX118.3. MCF-7 human breast carcinoma cells (ATCC HTB-22), HL-60 human leukemia cells (ATCC CCL-240), and EL4 mouse lymphoma cells (ATCC TIB-39) are suggested as positive controls. In immunohistochemistry, SX118 staining is primarily nuclear. Tonsil and colon are suggested as positive controls for tissue staining. In order to obtainn optimal indirect immunohistochemical staining, clone SXM30 should be titrated at 10-20 ug/ml and visualized via a three-step staining procedure in combination with a biotinylated polyclonal anti-mouse Ig (Cat. No. 550337, multiple absorbson) as the secondary antibody and Streptavidin-HRP (Cat. No. 550946) together with the DAB chromogen detection system (Cat. No. 550880). Refer to Fredersdorf et al., for information regarding induction of p21 expression, and for p21 distribution in different types of human tissues.
Store undiluted at 4°C. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
3. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
