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The pericentrin clone used is 1.7 kb in size and is derived from within residues 100-600 of mouse pericentrin 1. It was expressed as a fusion protein. The corresponding amino acids are present in both pericentrin and kendrin (pericentrin-2) so this antibody is predicted to cross-react with both isoforms.
Our Abpromise guarantee covers the use of ab4448 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||Use a concentration of 0.1 - 0.5 µg/ml.|
|ICC||Use at an assay dependent concentration.|
|IHC-FoFr||Use at an assay dependent concentration.|
ICC/IF image of ab4448 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab4448, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
ab4448 stained NIH-3T3 cells. The cells were 100% methanol fixed for 5 minutes at -20°C and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab4448 at 5µg/ml) overnight at +4°C. The secondary antibody (pseudo-colored green) was Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (ab150081) used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
NIH3T3 cells were fixed in 100% methanol for 6 minutes at -20°C, washed 3 times in PBS then incubated with ab4448 (1/2000) for 1 hour at room temperature. The panel of images shows the nuclei stained with DAPI (blue), ab4448 staining is shown in green. 100x magnification.
Paraffin embedded human kidney tissue sections were incubated with ab4448 (1/4500 dilution) for 30 minutes at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.
ab4448 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further images can be found at www.proteinatlas.org
IF staining of pericentrin in MCF7 cells (human).
The top panel is an interphase cell showing centrosome staining. The bottom panel shows a mitotic cell with spindle pole staining. ab4448 was used at 1/500, but also works at higher dilutions (1/1000-1/2000).
Top panel - 630X magnification; Bottom panel -1000X magnification. The secondary antibody was Alexa488 anti-rabbit.