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Western blotAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [5]
- Immunocytochemistry [1]
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- Product number
- MA5-15665 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- STAT5 beta Monoclonal Antibody (5B3)
- Antibody type
- Monoclonal
- Antigen
- Purifed from natural sources
- Description
- MA5-15665 targets STAT5B in WB applications and shows reactivity with Human samples.
- Antibody clone number
- 5B3
- Concentration
- Conc. Not Determined
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of STAT5B using STAT5B monoclonal antibody (Product # MA5-15665) in HeLa (1), K562 (2), NIH/3T3 (3), C6 (4), HEK293 (5), Jurkat (6) and HL-60 (7) cell lysate.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockout of STAT5B 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 STAT5B was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa Cas9 (Lane 2) and HeLa STAT5B 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 beta Monoclonal Antibody (5B3) (Product # MA5-15665, 1:1000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 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 STAT5B.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Knockdown of Signal transducer and activator of transcription 5B was achieved by transfecting HeLa with Signal transducer and activator of transcription 5B specific siRNAs (Silencer® select Product # s13539, s533811). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Signal transducer and activator of transcription 5B knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with STAT5 beta Monoclonal Antibody (5B3) (Product # MA5-15665, 1:1000 dilution ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Signal transducer and activator of transcription 5B.
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-STAT5 beta Monoclonal Antibody (5B3) (Product # MA5-15665) and a ~89kDa band corresponding to Signal transducer and activator of transcription 5B was observed across cell lines and tissues tested . Whole cell extracts (50 µg lysate) of HeLa (Lane 1), K-562 (Lane 2), THP-1 (Lane 3), MOLT-4 (Lane 4), Jurkat (Lane 5), NIH/3T3 (Lane 6), PC-12 (Lane 7), HEL 92.1.7 (Lane 8), Hep G2 (Lane 9), Mouse Lung (Lane 10), Rat Lung (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of STAT5B using STAT5B monoclonal antibody (Product # MA5-15665) in HeLa (1), K562 (2), NIH/3T3 (3), C6 (4), HEK293 (5), Jurkat (6) and HL-60 (7) cell lysate.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Signal transducer and activator of transcription 5B was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with STAT5 beta Monoclonal Antibody (5B3) (Product # MA5-15665) at 1:200 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Blue). Nuclei (Panel b:Green) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
