Subcellular distribution of Smn and hnRNP R in isolated mouse embryonic motoneurons.

Lentiviral knockdown of hnRNP R led to a dose-dependent reduction of hnRNP R levels. Calnexin and Smn protein were not altered significantly.

Primary motoneurons or E18 spinal cord tissue, respectively, were lysed with cytosolic and nuclear fractionation buffer, solubilized in Laemmli buffer and boiled for 10 minutes at 99°C. Proteins were then subjected to SDS-PAGE, blotted onto PVDF membrane, incubated with the corresponding antibodies, including ab22595.

 

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: empty lane
Lane 3: CANX knockout HAP1 whole cell lysate (20 µg)
Lane 4: empty lane
Lanes 1 - 4: Merged signal (red and green). Green - ab22595 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab22595 was shown to specifically react with CANX (Calnexin) in wildtype cells as signal was lost in CANX (Calnexin) knockout cells. Wild-type and eCANX (Calnexin) knockout samples were subjected to SDS-PAGE. ab22595 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1 dilution and 1/10,000 dilution respectively.

Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

ab22595 staining Calnexin in wild-type HAP1 cells (top panel) and CANX knockout HAP1 cells (bottom panel).

The cells were fixed with 4% formaldehyde for 10 minutes, 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 1 hour. The cells were then incubated with ab22595 at 1 μg/ml and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green).

Nuclear DNA was labeled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab22595 staining Calnexin in HeLa (Human epithelial cell line from cervix adenocarcinoma) cells.

The cells were fixed with 100% methanol for 5 minutes, 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 1 hour. The cells were then incubated with ab22595 at 1 μg/ml and ab7291 at 1 μg/ml overnight at +4°C, followed by a further incubation at room temperature for 1 hour with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor^® 594) (ab150120).

Nuclear DNA was labeled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: Calnexin knockout HAP1 cell lysate (20 µg)

Lanes 1 - 2: Merged signal (red and green). Green - ab22595 observed at 80 kDa. Red - loading control, ab8245, observed at 37 kDa.

This western blot image is a comparison between ab22595 and a competitor's top cited rabbit polyclonal antibody.

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

Lane 1 : NIH/3T3 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 : Mouse pancreas tissue lysate - total protein (ab29363)
Lane 8 : Testis (Mouse) Tissue Lysate - normal tissue (ab4027)
Lane 9 : Mouse skeletal muscle tissue lysate - total protein (ab29711)
Lane 10 : Spinal Cord (Mouse) Tissue Lysate (ab50253)
Lane 11 : Ovary (Mouse) Tissue Lysate (ab35808)
Lane 12 : PC-12 (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
All lanes : 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?

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

Lane 1 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
Lane 2 : U-2 OS (Human bone osteosarcoma epithelial cell line) whole cell lysate
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate
Lane 4 : HeLa whole cell lysate with Human Calnexin peptide (ab23379) at 1 µg/ml
Lane 5 : U-2 OS whole cell lysate with Human Calnexin peptide (ab23379) at 1 µg/ml
Lane 6 : MCF7 whole cell lysate with Human Calnexin peptide (ab23379) at 1 µg/ml

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : 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, U-2 OS and MCF7 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.

 

Calnexin was immunoprecipitated using 0.5 mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5 µg of Rabbit polyclonal to Calnexin - ER membrane marker and 50 µl of protein G magnetic beads (+).

No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.
Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70^oC; 10 µl 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.