Anti-STAT5a + STAT5b antibody [EPR16671-40]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Flow cytometry overlay histogram showing left K-562 positive cells and right negative PC3 stained with ab194898 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab194898) (1x 10⁶in 100μl at 0.04μg/ml (1/12500)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in K-562 Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling STAT5a + STAT5b with ab194898 at 1/250 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image showing both cytoplasmic and nuclear staining on K562 cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (AlexaFluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows :
1. ab194898 at 1/250 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Intracellular flow cytometric analysis of 2% paraformaldehyde-fixed K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling STAT5a +STAT5b with ab194898 at 1/80 dilution (red) compared with a rabbit monoclonal IgG isotype control (black) and a unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling STAT5a + STAT5b using ab194898 at 1/2000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 was used as secondary. Counter stained with Hematoxylin.
Inset image : negative control obtained using PBS instead of ab194898.
Nuclear and weak cytoplasmic staining on lymphocytes of human tonsil is observed.

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

• IP

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Immunoprecipitation - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

STAT5a + STAT5b were co-immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) cells with ab194898 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab178941 (Rabbit monoclonal [EPR16671] to STAT5b) at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, was used as secondary antibody at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898)

false

• IP

Supplier Data

Immunoprecipitation - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

STAT5a + STAT5b were co-immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) cells with ab194898 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab32043 (Rabbit monoclonal [E289] to STAT5a) at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, was used as secondary antibody at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898)

false

• IP

Supplier Data

Immunoprecipitation - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

STAT5a and STAT5b were immunoprecipitated from 1mg of K562 (Human chronic myelogenous leukemia cells from bone marrow) cells with ab194898 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab194898 at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated, was used as secondary antibody at 1/1000 dilution.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898)

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling STAT5a + STAT5b using ab194898 at 1/2000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 was used as secondary. Counter stained with Hematoxylin.
Inset image : negative control obtained using PBS instead of ab194898.
Nuclear and weak cytoplasmic staining on lymphocytes of rat spleen is observed.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling STAT5a + STAT5b using ab194898 at 1/2000 dilution. A Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 was used as secondary. Counter stained with Hematoxylin.
Inset image : negative control obtained using PBS instead of ab194898.
Nuclear and weak cytoplasmic staining on lymphocytes of mouse spleen is observed.

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

• WB

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Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/2000 dilution

Lane 1:

Human fetal heart lysates at 10 µg

Lane 2:

Human fetal kidney lysates at 10 µg

Secondary

All lanes:

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

false

• WB

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Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/20000 dilution

Lane 1:

K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysates at 20 µg

Lane 2:

Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysates at 20 µg

Lane 3:

Daudi (Human Burkitt's lymphoma cell line) whole cell lysates at 20 µg

Secondary

All lanes:

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

false

• WB

Supplier Data

Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Recombinant human C-terminal Myc/DDK tagged full length STAT5A from a commercial source.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/1000 dilution

All lanes:

Human STAT5A full length recombinant protein at 10 µg

Secondary

All lanes:

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

Predicted band size: 126 kDa,57 kDa

false

• WB

Supplier Data

Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (AB194898)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-STAT5a + STAT5b antibody [EPR16671-40] (ab194898) at 1/2000 dilution

Lane 1:

mouse heart lysates at 10 µg

Lane 2:

mouse kidney lysates at 10 µg

Lane 3:

mouse spleen lysates at 10 µg

Lane 4:

rat brain lysates at 10 µg

Lane 5:

rat heart lysates at 10 µg

Lane 6:

rat spleen lysates at 10 µg

Lane 7:

RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysates at 10 µg

Lane 8:

PC12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 10 µg

Lane 9:

NIH3T3 (Mouse embyro fibroblast cells) whole cell lysates at 10 µg

Secondary

All lanes:

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

false