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Hela cell fraction
References regarding specificity
Horner SM et al. Mitochondrial-associated endoplasmic reticulum membranes (MAM) form innate immune synapses and are targeted by hepatitis C virus. Proc Natl Acad Sci U S A 108:14590-5 (2011). PubMed: 21844353
Myhill N et al. The subcellular distribution of calnexin is mediated by PACS-2. Mol Biol Cell 19:2777-88 (2008). PubMed: 18417615
Yoshimura SI et al. Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus. J Cell Sci 114:4105-15 (2001). PubMed: 11739642
Our Abpromise guarantee covers the use of ab112995 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use at an assay dependent concentration.|
|In-Cell ELISA||Use a concentration of 1 µg/ml.|
|Flow Cyt||Use a concentration of 1 µg/ml. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.|
|ICC/IF||Use a concentration of 0.5 µg/ml.|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Calnexin knockout HAP1 cell lysate (20 µg)
Lane 3: THP1 cell lysate (20 µg)
Lane 4: Raw264.7 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab112995 observed at 80 kDa. Red - loading control, ab181602, observed at 37 kDa.
ab112995 was shown to specifically react with Calnexin when Calnexin knockout samples were used. Wild-type and Calnexin knockout samples were subjected to SDS-PAGE. ab112995 at a concentration of 1 µg/mL and ab181602 (loading control to GAPDH) diluted to 1/1000 were incubated overnight at 4°C. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (ab216777) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.
ab112995 staining Calnexin in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab112995 at 0.5μg/ml and ab202272 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Mouse IgG (Alexa Fluor® 488) (ab150117) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).
ab112995 has not yet been referenced specifically in any publications.
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