All lanes : Anti-STAT6 antibody [YE361] (ab32520) at 1/1000 dilution (purified)

Lane 1 : NIH/3T3 (mouse embyro fibroblast cell line) cell lysate
Lane 2 : RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunohistochemical staining of paraffin embedded human kidney with purified ab32520 at a working dilution of 1/50. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

Flow Cytometry analysis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling STAT6 with purified ab32520 at 1/30 dilution(10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor® 488)(1:2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.

Immunofluorescence staining of HeLa (human epithelial cell line from cervix adenocarcinoma) cells with purified ab32520 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab32520 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

ab32520 (purified) at 1/20 immunoprecipitating STAT6 in 10 μg NIH/3T3 (mouse embyro fibroblast cell line; Lanes 1 and 2, observed at 100 kDa). Lane 3 - PBS. For western blotting, HRP Veriblot for IP (ab131366) was used for detection (1/10 000). Blocking buffer and concentration: 5% NFDM/TBST Dilution buffer and concentration: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

Anti-STAT6 antibody [YE361] (ab32520) at 1/20000 dilution (purified) + Raji (human Burkitt's lymphoma cell line) cell lysate at 10 µg

Secondary
HRP goat anti-rabbit IgG (H+L) at 1/20000 dilution

Predicted band size: 94 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human skin carcinoma tissue labelling STAT6 with unpurified ab32520 at 1/100.

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

Immunocytochemistry/Immunofluorescence analyis of HeLa (human epithelial cell line from cervix adenocarcinoma) cells labelling STAT6 with unpurified ab32520 at 1/100.

Anti-STAT6 antibody [YE361] (ab32520) at 1/1000 dilution (unpurified) + NIH/3T3 (mouse embyro fibroblast cell line) cell lysate

Predicted band size: 94 kDa
Observed band size: 100 kDa why is the actual band size different from the predicted?

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human transitional cell carcinoma of bladder tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human glioma tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human solitary fibrous tumor tissue sections labelling STAT6 with unpurified ab32520 at a dilution of 1/1000. HRP goat anti-rabbit (ab97051) was used at a dilution of 1/500. The antigen retrieval solution was Tris-EDTA buffer, pH 9.0.