Anti-galectin 9/Gal-9 antibody [EPR22214]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

Intracellular 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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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).

• WB

Lab

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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.

All lanes:

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/500 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Galectin 9 knockout A549 cell lysate at 20 µg

Lane 2:

Western blot - Human Lgals9 (galectin 9/Gal-9) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-lgals9-galectin-9-gal-9-knockout-a549-cell-line-ab266923'>ab266923</a>)

Lane 3:

U-937 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 40 kDa

Observed band size: 34-39 kDa

false

• WB

Supplier Data

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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).

All lanes:

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg

Lane 2:

HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 40 kDa

Observed band size: 13-15 kDa,34-39 kDa

false

Exposure time: 59s

• WB

Supplier Data

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

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.

All lanes:

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution

Lane 1:

U937 (human histiocytic lymphoma monocyte), whole cell lysate at 20 µg

Lane 2:

HL-60 (human Acute Promyelocytic Leukemia promyeloblast), whole cell lysate at 20 µg

Lane 3:

MOLT-4 (human lymphoblastic leukemia T lymphoblast), whole cell lysate at 20 µg

Lane 4:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 40 kDa

Observed band size: 13-15 kDa,34-39 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (AB227046)

Lanes 1 - 2 : Merged signal (red and green). Green - ab227046 observed at 35 kDa. Red - loading control Mouse anti Histone H3 observed at 18 kDa.

ab227046 was shown to react with galectin 9/Gal-9 in wild-type THP-1 cells in Western blot with loss of signal observed in LGALS9 knockout cell line ab269505 (knockout cell lysate ab269667). Wild-type THP-1 and LGALS9 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab227046 and Mouse anti Histone H3 overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-galectin 9/Gal-9 antibody [EPR22214] (ab227046) at 1/1000 dilution

Lane 1:

Wild-type THP-1 cell lysate at 20 µg

Lane 2:

LGALS9 knockout THP-1 cell lysate at 20 µg

Lane 2:

Western blot - Human Lgals9 (galectin 9/Gal-9) knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-lgals9-galectin-9-gal-9-knockout-thp-1-cell-line-ab269505'>ab269505</a>)

Predicted band size: 40 kDa

Observed band size: 35 kDa

false