All lanes : Anti-galectin 9/Gal-9 antibody [EPR23853-132] (ab275877) at 1/1000 dilution

Lane 1 : Mouse thymus tissue lysate
Lane 2 : Mouse liver tissue lysate
Lane 3 : Mouse lung tissue lysate
Lane 4 : Mouse brain tissue lysate
Lane 5 : EL4.IL-2 (mouse lymphoma t lymphocyte) whole cell lysate
Lane 6 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 7 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lysates/proteins at 40 µg per lane.

Secondary
Lanes 1-4 : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/50000 dilution
Lanes 5-7 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 40 kDa
Observed band size: 15,36,40 kDa why is the actual band size different from the predicted?

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

Bands of 40KDa and 36KDa respectively represent two different isoforms.

Band of 15KDa may represents the cleaved form.

The molecular weight observed is consistent with what has been described in the literature (PMID: 9038233, 11886844, 22805533, 15811318).

Low expression: mouse brain (PMID: 9038233).

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time: Lanes 1-2: 5.5 seconds; Lanes 3-6: 37 seconds; Lanes 7: 3 minutes.

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized EL4.IL-2 cells labelling galectin 9/Gal-9 with ab275877 at 1/50 (12.28 µg/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing membranous and cytoplasmic staining in EL4.IL-2 cell line. The staining pattern observed is consistent with what has been described in the literature (PMID: 28750861). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - 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: Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized EL4.IL-2 (mouse lymphoma T lymphocyte) cells labelling galectin 9/Gal-9 with ab275877 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) 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.

Anti-galectin 9/Gal-9 antibody [EPR23853-132] (ab275877) at 1/1000 dilution + Recombinant mouse galectin 9/Gal-9 protein, 20 ng

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 40 kDa

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

Exposure time: 15 seconds.