This antibody detects a 19kD band in Hela and HEK293 cells. The band can be blocked with the immunising peptide.
The antibody shows extensive staining (eg. of lymphoid and epithelial cells), with staining of nuclei and some staining of the cytoplasm, when tested on human tonsil in IHC.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/500. Detects a band of approximately 19 kDa (predicted molecular weight: 18.5 kDa).
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml.
Essential component of the SCF (SKP1-CUL1-F-box protein) ubiquitin ligase complex, which mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction and transcription. In the SCF complex, serves as an adapter that links the F-box protein to CUL1. SCF(BTRC) mediates the ubiquitination of NFKBIA at 'Lys-21' and 'Lys-22'; the degradation frees the associated NFKB1-RELA dimer to translocate into the nucleus and to activate transcription. SCF(Cyclin F) directs ubiquitination of CP110.
ICC/IF image of ab10546 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab10546, 1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Western blot - Anti-Skp1 antibody (ab10546)
Western blot using ab10546 at 1/500.
Lane 1: Hela nuclear Lane 2: Hela Lane 3: HEK293 Lane 4: Hela nuclear + ab10546 blocking peptide (1µg/ml) Lane 5: Hela + ab10546 blocking peptide (1µg/ml) Lane 6: HEK293 + ab10546 blocking peptide (1µg/ml)
ab10546 was used in immunohistochemistry with paraffin embedded sections of human tonsil, using DAB as a chromogen (brown). Counterstaining of nuclei was performed with haemotoxylin (blue). Strong staining of epithelial cell nuclei is seen (and some staining of the cytoplasm) in this image.