Immunocytochemistry/ Immunofluorescence - Calnexin antibody (ab22595)

ICC/IF image of ab22595 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab22595, 5µg/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-iT^TM 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).

Western blot - Calnexin antibody (ab22595)

All lanes : Anti-Calnexin - ER membrane marker antibody (ab22595) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line)
Lane 2 : U2OS Whole Cell Lysate
Lane 3 : MCF-7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lane 4 : HeLa (Human epithelial carcinoma cell line) with Calnexin peptide (ab23379) at 1 µg/ml
Lane 5 : U2OS Whole Cell Lysate with Calnexin peptide (ab23379) at 1 µg/ml
Lane 6 : MCF-7 (Human breast adenocarcinoma cell line) Whole Cell Lysate with Calnexin peptide (ab23379) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG (Alexa Fluor® 680) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 90 kDa
Observed band size : 75 kDa (why is the actual band size different from the predicted?)

Recent batches of ab22595 (AP217379 and AP151845) detect a band of ~ 75 kDa in Hela, U2OS and MCF-7 lysates. This band is completely blocked by the immunizing peptide so we believe this represents Calnexin. Moreoever, a band of the same size is detected by other Calnexin antibodies tested.

Immunoprecipitation - Anti-Calnexin - ER membrane marker antibody (ab22595)

Calnexin - ER membrane marker was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Calnexin - ER membrane marker and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab22595.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
Band: 80kDa: Calnexin - ER membrane marker.

Western blot - Calnexin - ER membrane marker antibody (ab22595)

All lanes : Anti-Calnexin - ER membrane marker antibody (ab22595) at 1/250 dilution

Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab7179)
Lane 2 : MEF1 (Mouse embryonic fibroblast cell line) Whole Cell Lysate (ab46770)
Lane 3 : Brain (Mouse) Tissue Lysate (ab27253)
Lane 4 : Liver (Mouse) Tissue Lysate (ab7935)
Lane 5 : Heart (Mouse) Tissue Lysate (ab27255)
Lane 6 : Kidney (Mouse) Tissue Lysate (ab27254)
Lane 7 : Pancreas (Mouse) Tissue Lysate (ab29363)
Lane 8 : Testis (Mouse) Tissue Lysate - normal tissue (ab4027)
Lane 9 : Skeletal Muscle (Mouse) Tissue Lysate (ab29711)
Lane 10 : Spinal Cord (Mouse) Tissue Lysate (ab50253)
Lane 11 : Ovary (Mouse) Tissue Lysate (ab35808)
Lane 12 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957)
Lane 13 : Brain (Rat) Tissue Lysate (ab7942)
Lane 14 : Liver (Rat) Tissue Lysate (ab27256)
Lane 15 : Heart (Rat) Tissue Lysate (ab7940)
Lane 16 : Kidney (Rat) Whole Cell Lysate - normal tissue (ab29480)

Lysates/proteins at 10 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 90 kDa
Observed band size : 80 kDa (why is the actual band size different from the predicted?)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Calnexin - ER membrane marker antibody (ab22595)

Kidney cortex using ab22595 shows clear cytoplasmic staining patterns. The visceral cells of the Glomerular tuft ( podocytes ) are strongly stained (indicated by red arrowheads). Distal convoluted tubular cells are generally moderately positive (with exceptions that are strongly positive). However, most of the cells that line the Proximal Convoluted Tubules (indicated by green arrowheads) are strongly positive.

Carl Hobbs, King`s College London, United Kingdom

Immunocytochemistry/ Immunofluorescence - Calnexin - ER membrane marker antibody (ab22595)

ICC/IF image of ab22595 stained HeLa cells. The cells were 4% PFA 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 (ab22595, 5µg/ml) overnight at +4ºC. The secondary antibody (green) was DyLight® 488 goat anti-rabbit IgG - H&L, pre-adsorbed (ab96899) 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.