Anti-Galectin 8/Gal-8 antibody [EPR4857] (ab109519) at 1/10000 dilution (purified) + LNCAP (human prostat carcinoma) whole cell lysate at 10 µg

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

Predicted band size: 40 kDa
Observed band size: 40 kDa



Blocking and diluting buffer 5% NFDM/TBST

ab109519 staining Galectin 8/Gal-8 in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab7291 and ab150120 were used as counterstains for primary antibody ab109519 and secondary antibody ab150077 respectively and DAPI was used as a nuclear counterstain.

Negative control 1: Rabbit primary antibody and anti-mouse secondary antibody (ab150120)
Negative control 2: Mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077)

ab109519 staining Galectin 8/Gal-8 in the human cell line HEK293 (human embryonic kidney) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/100. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/500 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

Unlabelled control: Cell without incubation with primary antibody and secondary antibody (Blue)

Purified ab109519 at 1/30 dilution (2µg) immunoprecipitating Galectin 8/Gal-8 in HeLa whole cell lysate.
Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10µg
Lane 2 (+): ab109519 + HeLa whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109519 in HeLa whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/10,000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 36/40 kDa

All lanes : Anti-Galectin 8/Gal-8 antibody [EPR4857] (ab109519) at 1/1000 dilution (unpurified)

Lane 1 : HepG2 cell lysate
Lane 2 : LNCaP cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 40 kDa

Lanes 1 & 3 : unpurified at 1/1000 dilution
Lane 2 : Anti-Galectin 8/Gal-8 antibody [EPR4857] (ab109519) at 1/200 dilution (unpurified)

All lanes : Galectin 9 recombinant protein

Lysates/proteins at 0.02 µg per lane.

Predicted band size: 40 kDa

ab109519 has weak cross-reactivity with Galectin 9.

ab109519 staining Galectin 8/Gal-8 in human prostatic carcinoma tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/250. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

Negative control 1: PBS in place of primary antibody.

Unpurified ab109519, at a 1/100 dilution, staining Human prostatic adenocarcinoma, using Immunohistochemstry, Formalin/PFA-fixed paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing HeLa cells stained with unpurified ab109519 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab109519, 1/100) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.