Purification notesab112995 was produced in vitro using hybridomas grown in serum-free medium, and then purified by biochemical fractionation. Monoclonal purity was near homogeneity as judged by SDS-PAGE.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
Use a concentration of 0.5 µg/ml.
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
FunctionCalcium-binding protein that interacts with newly synthesized glycoproteins in the endoplasmic reticulum. It may act in assisting protein assembly and/or in the retention within the ER of unassembled protein subunits. It seems to play a major role in the quality control apparatus of the ER by the retention of incorrectly folded proteins.
Sequence similaritiesBelongs to the calreticulin family.
Cellular localizationEndoplasmic reticulum membrane. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
Histocompatibility complex class I antigen binding protein p88 antibody
Major histocompatibility complex class I antigen-binding protein p88 antibody
Anti-Calnexin antibody [6F12BE10] images
Western blot - Anti-Calnexin antibody [6F12BE10] (ab112995)
Additional bands at : 80 kDa. We are unsure as to the identity of these extra bands.
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. 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 (shown in green) in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated with ab112995 at 0.5 μg/ml and ab202272 at 1/250 dilution (alpha tubulin shown in red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour 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).
Immunoprecipitation with ab112995.
Immunoprecipitation of Calnexin - ER membrane marker from HeLa cell lysate. The protein band runs around 90 kDa (predicted 68kDa) in tris-glycine SDS-PAGE. The identity of this protein was confirmed by mass spectrometry. This gel was stained with colloidal Coomassie blue G.
ab112995 at 5µg/ml staining Calnexin - ER membrane marker in Human fibroblasts cells by Immunocytochemistry (4% paraformaldehyde fixed and 0.1% Triton X-100 permeabilized) followed by Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h(green).
IHC image of ab112995 staining in human colon formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab112995, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
References for Anti-Calnexin antibody [6F12BE10] (ab112995)
has not yet been referenced specifically in any publications.
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