Anti-STAT5b antibody [EPR16671] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

Intracellular 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

ab178941 staining STAT5b in the human cell line HeLa (human cervix adenocarcinoma) by intracellular 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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

• IP

Lab

Immunoprecipitation - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

This data was developed using the same antibody clone in a different buffer formulation (ab178941)

Immunoprecipitation of STAT5B in HAP1 cells. Lysates were prepared and immunoprecipitation was performed using 2.0 µg of ab178941 pre-coupled to Protein A beads. Samples were washed and processed for western blot with STAT5 beta Monoclonal Antibody at 1/200.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

STAT5 beta Monoclonal Antibody at 1/200 dilution

All lanes:

HAP1 cells

Observed band size: 89 kDa

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• ChIP

Unknown

ChIP - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

*http : //www.abcam.com/resources?keywords=X%20ChIP%20protocol

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

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-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

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

• WB

Lab

Western blot - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

This data was developed using the same antibody clone in a different buffer formulation (ab178941)

ab178941 was shown to react with STAT5B in wild-type HAP1 cells in Western blot with loss of signal observed in a STAT5B knockout cell line. Wild-type HAP1 and STAT5B knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab178941 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-STAT5b antibody [EPR16671] (<a href='/en-us/products/primary-antibodies/stat5b-antibody-epr16671-ab178941'>ab178941</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 lysate at 20 µg

Lane 2:

STAT5B knock-out HAP1 lysate at 20 µg

Observed band size: 89 kDa

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• IP

Supplier Data

Immunoprecipitation - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

All lanes:

Immunoprecipitation - Anti-STAT5b antibody [EPR16671] (<a href='/en-us/products/primary-antibodies/stat5b-antibody-epr16671-ab178941'>ab178941</a>)

Predicted band size: 89 kDa

Observed band size: 90 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab178941).

All lanes:

Immunoprecipitation - Anti-STAT5b antibody [EPR16671] (<a href='/en-us/products/primary-antibodies/stat5b-antibody-epr16671-ab178941'>ab178941</a>)

Predicted band size: 89 kDa

false

• WB

Lab

Western blot - Anti-STAT5b antibody [EPR16671] - BSA and Azide free (AB240211)

This data was developed using the same antibody clone in a different buffer formulation (ab178941).

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.

All lanes:

Western blot - Anti-STAT5b antibody [EPR16671] (<a href='/en-us/products/primary-antibodies/stat5b-antibody-epr16671-ab178941'>ab178941</a>) at 1/20000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

STAT5B knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human STAT5B knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-stat5b-knockout-hela-cell-line-ab266006'>ab266006</a>)

Predicted band size: 89 kDa

Observed band size: 90 kDa

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