All lanes : Anti-YB1 antibody [EPR22682-21] (ab255606) at 1/1000 dilution

Lane 1 : RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 36 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

The molecular weight observed is consistent with what has been described in the literature (PMID:29262622).

Blocking/diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 7.75 seconds

All lanes : Anti-YB1 antibody [EPR22682-21] (ab255606) at 1/1000 dilution

Lane 1 : Human tonsil tissue lysate
Lane 2 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
Lane 1 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution
Lanes 2-3 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?

Blocking/diluting buffer and concentration: 5% NFDM/TBST

The molecular weight observed is consistent with what has been described in the literature (PMID:29262622).

Exposure time:

Lane 1: 10 seconds

Lanes 2-3: 37 seconds

YB1 was immunoprecipitated from 0.35 mg MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10μg with ab255606 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab255606 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1/5000 dilution.

Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2: ab255606 IP in MCF7 whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab255606 in MCF7 whole cell lysate.

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

Exposure time: 30 seconds

Immunohistochemical analysis of paraffin-embedded Human mammary gland tissue labeling YB1 with ab255606 at 1/4000 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in human normal mammary gland (PMID: 21695211) is observed.  Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling YB1 with ab255606 at 1/4000 dilution  followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining in tumor cells of human breast cancer (PMID: 21695211) is observed. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling YB1 with ab255606 at 1/100 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in NIH/3T3 cell line is observed. ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)  was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized MCF7 (human breast adenocarcinoma epithelial cell) cells labelling YB1 with ab255606 at 1/100 dilution, followed by ab150077 AlexaFluor®488 Goat anti-Rabbit secondary antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in MCF7 cell line is observed. ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized MCF7 (Human breast adenocarcinoma epithelial cell) cells labelling YB1 with ab255606 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.  

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A549 (Human lung carcinoma epithelial cell) cells labelling YB1 with ab255606 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730, Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.