Immunocytochemistry analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Cell line U937 (human histiocytic lymphoma cell line) cells labeling galectin 9/Gal-9 with ab227046 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green).
The nuclear counterstain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at a 1/200 dilution (red).

Confocal image showing nuclear and cytoplasmic staining in U937 cell line.

Negative control: PANC-1.

All lanes : Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/500 dilution

Lane 1 : Wild-type A549 cell lysate
Lane 2 : Galectin 9 knockout A549 cell lysate
Lane 3 : U-937 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 40 kDa
Observed band size: 34-39 kDa why is the actual band size different from the predicted?

Lanes 1-4: Merged signal (red and green). Green - ab227046 observed at 34-39 kDa. Red - loading control ab8245 observed at 36 kDa.

ab227046 Anti-galectin 9/Gal-9 antibody [EPR22214] was shown to specifically react with galectin 9/Gal-9 in wild-type A549 cells. Loss of signal was observed when knockout cell line ab266923 (knockout cell lysate ab256976) was used. Wild-type and galectin 9/Gal-9 knockout samples were subjected to SDS-PAGE. ab227046 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 500 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling galectin 9/Gal-9 with ab227046 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear and cytoplasmic staining on Kupffer cells of human liver (PMID: 20209097, PMID: 18202194) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution

Lane 1 : U937 (human histiocytic lymphoma monocyte), whole cell lysate
Lane 2 : HL-60 (human Acute Promyelocytic Leukemia promyeloblast), whole cell lysate
Lane 3 : MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate
Lane 4 : PANC-1 (human pancreatic epithelioid carcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 40 kDa
Observed band size: 34-39 kDa why is the actual band size different from the predicted?
Additional bands at: 13-15 kDa (possible degradation product)


Exposure time: 59 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression of the isoforms observed is consistent with what has been described in the literature (PMID 11886844, PMID: 22805533).

Degraded fragments (13-15 kDa) have also been descried (PMID: 15811318).

Negative control: PANC-1.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized PANC-1 (human pancreatic epithelial cancinoma cell line) (Left) / U937 (human histiocytic lymphoma cell line) (Right) cell line labeling galectin 9/Gal-9 with ab227046 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

Negative Control - PANC-1 cells.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling galectin 9/Gal-9 with ab227046 at 1/1000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear and cytoplasmic staining on epithelial cells of human colon (PMID: 18202194) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney) cells labeling galectin 9/Gal-9 with ab227046 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing no staining in HEK-293T cells

Negative control: HEK-293T (PMID: 11698107). The nuclear counterstain is DAPI (blue).
Counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).
Secondary antibody only control: Used PBS instead of the primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution

Lane 1 : THP-1 (human monocytic leukemia monocyte), whole cell lysate
Lane 2 : HEK-293T (human embryonic kidney epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 40 kDa
Observed band size: 34-39 kDa why is the actual band size different from the predicted?
Additional bands at: 13-15 kDa (possible degradation product)


Exposure time: 59 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The expression of the isoforms observed is consistent with what has been described in the literature (PMID 11886844, PMID: 22805533).

Degraded fragments (13-15 kDa) has also been described (PMID: 15811318).

Negative control: HEK-293T (PMID: 24333696).