- 33-5900 - Invitrogen Antibodies STAT5B antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of K562 cell lysates using Ms anti-STAT5 (Pan) (Product # 33-5900).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), K562 (Lane 2), HEL 92.1.7 (Lane 3) and Hep G2 (Lane 4). The blots were probed with Anti-STAT5 Mouse Monoclonal Antibody (Product # 33-5900, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A 86 kDa band corresponding to STAT5 was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a transferred onto a nitrocellulose membrane with Pierce™ Power Blotter System (22834). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of STAT5 was achieved by transfecting HeLa cells with STAT5 specific siRNAs (Silencer® select Product # s1353 and s13536). Western blot analysis (Fig a) was performed using Whole cell extract from the STAT5 knock down cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-STAT5 Mouse Monoclonal Antibody (Product # 33-5900, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to STAT5.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Whole cell extracts (30 µg lysate) of HeLa (Lane 1), K-562 (Lane 2), THP-1 (Lane 3), MOLT-4 (Lane 4) and Jurkat (Lane 5). The blot was probed with Anti-STAT5 Mouse Monoclonal Antibody (Product # 33-5900, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 90 kDa band corresponding to STAT5 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of STAT5 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR773180_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of STAT5 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) andHeLa STAT5 KO (Lane 3) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-STAT5 alpha/beta Monoclonal Antibody (ST5-8F7) (Product # 33-5900, 1:500 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:8,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to STAT5. An uncharacterized band was oberved at ~40 kDa in all the samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of STAT5 was performed using 0.7 % confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with STAT5 Mouse Monoclonal antibody (Product # 33-5900) at 5 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear and Cytoplasm localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Supportive validation

33-5900