All lanes : Anti-Aldolase + Aldolase C antibody [EPR19355] (ab200049) at 1/1000 dilution

Lane 1 : Human Aldolase C full length recombinant protein
Lane 2 : Human Aldolase full length recombinant protein
Lane 3 : Human Aldolase B full length recombinant protein

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 39 kDa
Observed band size: 39 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Human ALDOC full length recombinant protein contains aa2-364 with a His-Tag®. Human ALDOA and ALDOB full length recombinant protein contains aa1-364 with a His-Tag®. These three recombinant proteins were made in-house.

This product showed very weak reactivity to Aldolase B, relative to Aldolase and Aldolase C, as demonstrated by WB of recombinant proteins.

Lanes 1-3 : Anti-Aldolase + Aldolase C antibody [EPR19355] (ab200049) at 1/5000 dilution
Lanes 4-7 : Anti-Aldolase + Aldolase C antibody [EPR19355] (ab200049) at 1/2000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : U 87 MG (Human glioblastoma-astrocytoma epithelial cell line) whole cell lysate
Lane 3 : Human cerebellum lysate
Lane 4 : Human fetal liver lysate
Lane 5 : Human fetal heart lysate
Lane 6 : Human fetal kidney lysate
Lane 7 : human fetal spleenlysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lanes 4-7 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 39 kDa
Additional bands at: 39 kDa (possible non-specific secondary antibody binding)

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-3: 8 seconds; Lane 4-7: 10 seconds.

The expression profile observed is consistent with what has been described in the literature (PMID:  24475166 and 17659271).

Lanes 1-6 : Anti-Aldolase + Aldolase C antibody [EPR19355] (ab200049) at 1/5000 dilution
Lane 7 : Anti-Aldolase + Aldolase C antibody [EPR19355] (ab200049) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : Rat brain lysate
Lane 3 : Rat kidney lysate
Lane 4 : Mouse heart lysate
Lane 5 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 6 : Mouse brain lysate
Lane 7 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 39 kDa
Observed band size: 39 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-5: 8 seconds; Lane 6/7: 3 seconds.

 

Immunofluorescent analysis of 100% methanol-fixed U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling Aldolase C + Aldolase with ab200049 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on U-87 MG cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab200049 at 1/500 dilution followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Immunofluorescent analysis of 100% methanol-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Aldolase C + Aldolase with ab200049 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab200049 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed U-87 MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling Aldolase + Aldolase C with ab200049 at 1/600 dilution (red) compared with a rabbit monoclonal IgG isotype control (ab172730; black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (Alexa Fluor^® 488) at 1/500 dilution was used as the secondary antibody.