All lanes : Anti-STAT5b antibody [EPR16671] (ab178941) at 1/20000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : STAT5B knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 90 kDa
Observed band size: 90 kDa

Lanes 1-2: Merged signal (red and green). Green - ab178941 observed at 90 kDa. Red - loading control ab8245 observed at 37 kDa.

 ab178941 Anti-STAT5b antibody [EPR16671] was shown to specifically react with STAT5b in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab266006 (knockout cell lysate ab257710) was used. Wild-type and STAT5b knockout samples were subjected to SDS-PAGE. ab178941 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 20000 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.

 

Immunoprecipitation of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell extract using ab178941 at 1/40 dilution. Western blot detection of STAT5b utilised ab178941 at 1/2000 dilution and Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at 1/1000 dilution. The blocking and dilution buffer was 5% NFDM/TBST.

Chromatin was prepared from T-47D (starved overnight) treated with Prolactin(10nM 30min) and T-47D(starved overnight) non-treated cells according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30mins and then formaldehyde for 10min.
The ChIP was performed with 25 μg of chromatin, 5 μg of ab178941 (red), or 5 μg of rabbit normal IgG ab172730 (gray) and 20 μL of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
Primers are from PMID: 15686596.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling STAT5b with ab178941 at 1/100 dilution. The cells were permeabilised with 0.1% Triton X-100. Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) at 1/200 dilution was used as the secondary antibody (green). Nuclear and cytoplasm staining is detected. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (Tubulin mouse mAb) at 1/500 and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/400 dilution (red). 

 

The negative controls are as follows;

1. ab178941 at 1/100 dilution followed by Goat anti mouse IgG (Alexa Fluor®594) at 1/400 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by Goat anti rabbit IgG (Alexa Fluor®488) ar 1/200 dilution.

 

 

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling STAT5b with ab178941 at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus staining on lymphocytes of Human spleen is detected. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

ab178941 staining STAT5b in the human cell line HeLa (human cervix adenocarcinoma) by flow cytometry. Cells were fixed with 4% paraformaldehyde and the sample was incubated with the primary antibody at a dilution of 1/40. A goat anti rabbit IgG (Alexa Fluor® 488) at a dilution of 1/2000 was used as the secondary antibody.

Isoytype control: Rabbit monoclonal IgG (Black)

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

All lanes : Anti-STAT5b antibody [EPR16671] (ab178941) at 1/20000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysates
Lane 3 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates
Lane 4 : Daudi (Human Burkitt's lymphoma cell line) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 90 kDa
Observed band size: 90 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-STAT5b antibody [EPR16671] (ab178941) at 1/5000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Mouse heart lysates
Lane 3 : Mouse kidney lysates
Lane 4 : Mouse spleen lysates
Lane 5 : Rat brain lysates
Lane 6 : Rat heart lysates
Lane 7 : Rat kidney lysates
Lane 8 : Rat spleen lysates
Lane 9 : C6 (Rat glial tumor cells) whole cell lysates
Lane 10 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates
Lane 11 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates
Lane 12 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysates

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 90 kDa
Observed band size: 90 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

All lanes : Anti-STAT5b antibody [EPR16671] (ab178941) at 1/20000 dilution

Lane 1 : Human fetal heart lysates
Lane 2 : Human fetal kidney lysates
Lane 3 : Human fetal spleen lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 90 kDa
Observed band size: 90 kDa

Blocking/dilution buffer: 5% NFDM/TBST.

Lane 1 : Anti-STAT5b antibody [EPR16671] (ab178941) at 1/1000 dilution
Lane 2 : Anti-STAT5a antibody [E289] (ab32043) at 1/5000 dilution

All lanes : STAT5a recombinant protein

Developed using the ECL technique.

Predicted band size: 90 kDa

WB showing no cross reactivity with STAT5a.

Cross Immunoprecipitation of K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell extract showing no cross reactivity with STAT5a. Protein captured by anti-STAT5a antibody (ab32042) was detected by the same antibody in WB (image 1) but not by anti-STAT5b, ab178941 (image 2).

 

For WB detection, ab178941 was used at a 1/2000 dilution and Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated secondary antibody at a 1/1000 dilution. The blocking and dilution buffer was 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling STAT5b with ab178941 at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus staining on lymphocytes of Mouse spleen is detected. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labeling STAT5b with ab178941 at 1/500 dilution, followed by prediluted HRP Polymer for Rabbit/Mouse IgG. Nucleus staining on lymphocytes and weak nucleus staining on gland epithelium of colon is detected. The negative control utilised PBS insead of primary antibody and the slide is counter stained with Hematoxylin.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow cytometry analysis of 2% paraformaldehyde K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling STAT5b with ab178941 at 1:60 dilution (red line). Secondary antibody used is a goat anti rabbit IgG (FITC) at 1:150 dilution. The isotype control is rabbit monoclonal IgG (black line).