All lanes : Anti-Cdc34 antibody [EPR16808] (ab204515) at 1/5000 dilution

Lane 1 : C6 (Rat glial tumor cells) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 26 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 2 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Neuro-2a (Mouse neuroblastoma cells) cells labeling Cdc34 with ab204515 at 1/50 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on Neuro-2a cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab204515 at 1/50 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed Jurkat (Human T cell leukemia cells from peripheral blood) cells labeling Cdc34 with ab204515 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

All lanes : Anti-Cdc34 antibody [EPR16808] (ab204515) at 1/5000 dilution

Lane 1 : Recombinant protein fragment human Cdc34
Lane 2 : Recombinant protein fragment human Cdc34B

Lysates/proteins at 0.01 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 26 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 1 second

All lanes : Anti-BTK (phospho Y223) antibody [EP420Y] (Alexa Fluor® 488) (ab204516) at 1/5000 dilution

Lane 1 : 293 (Human epithelial cells from embryonic kidney) whole cell lysate
Lane 2 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/100000 dilution

Predicted band size: 26 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 5 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Cdc34 antibody [EPR16808] (ab204515) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Mouse kidney lysate
Lane 4 : Rat heart lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 26 kDa
Observed band size: 35 kDa why is the actual band size different from the predicted?


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Cdc34 with ab204515 at 1/50 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear and cytoplasmic staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab204515 at 1/50 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Cdc34 with ab204515 at 1/100 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/500 dilution was used as the secondary antibody.

Cdc34 was immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate with ab204515 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab204515 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: K562 whole cell lysate 10ug (Input).

Lane 2: ab204515 IP in K562 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab204515 in K562 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 5 seconds.