701430
antibody from Invitrogen Antibodies
Targeting: NFKB1
KBF1, NF-kappaB, NF-kB1, NFkappaB, NFKB-p50, p105, p50
Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Flow cytometry [1]
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Validation data
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- Product number
- 701430 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Phospho-NFkB p50 (Ser337) Recombinant Rabbit Monoclonal Antibody (2H7L8)
- Antibody type
- Monoclonal
- Antigen
- Other
- Description
- 701430 has been successfully used in Western blot, flow cytometry, and immunofluorescence applications.
- Antibody clone number
- 2H7L8
- Concentration
- 0.5 mg/mL
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of p50 (pS337) was performed by loading 20 µg of Jurkat, Jurkat treated with TNF-alpha (20 ng/mL for 30 minutes) lysates (lane 1, 2) using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. p50 (pS337)was detected at ~50 kDa using p50 (pS337) Recombinant Rabbit Monoclonal Antibody (Product # 701430) at 1:1000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. To confirm specificity, competition was performed with the phosphopeptide (10 µg/mL) (lane 3, 4). Goat anti-Rabbit IgG-HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescent analysis of p-NFkBp50 Ser337 (green) in A549 cells untreated and 20 ng/mL TNF-alpha treated cells for 20 hours. The cells were fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at room temperature. Cells were stained with a p-NFkBp50 Recombinant Rabbit Monoclonal Antibody (Product # 701430) at a dilution of 1:400 in blocking buffer overnight at 4C, and then incubated with a Goat anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor Plus 488 conjugate (Product # A32731) at a dilution of 1:500 for at least 30 minutes at a room temperature in the dark (green). Nuclei (blue) were stained with Hoechst 33342 (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow cytometry analysis of p50 [pS337] was done on Jurkat cells treated with TNF alpha (20 ng/ml, 30 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª p50 [pS337] Recombinant Rabbit Monoclonal Antibody (701430, red histogram) or with rabbit isotype control (pink histogram) at a dilution of 1:400 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor¨ 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.