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Western blotAntibody data
- Antibody Data
- Antigen structure
- References [1]
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
- Flow cytometry [1]
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- Product number
- PA1-830 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- c-Fos Polyclonal Antibody
- Antibody type
- Polyclonal
- Antigen
- Synthetic peptide
- Description
- PA1-830 detects cFos from mouse and human cells.
- Concentration
- 1 mg/mL
Submitted references 25-Hydroxycholesterol acts as an amplifier of inflammatory signaling.
Gold ES, Diercks AH, Podolsky I, Podyminogin RL, Askovich PS, Treuting PM, Aderem A
Proceedings of the National Academy of Sciences of the United States of America 2014 Jul 22;111(29):10666-71
Proceedings of the National Academy of Sciences of the United States of America 2014 Jul 22;111(29):10666-71
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of c-Fos (Product # PA1-830) was performed by loading 50 µg of Serum (20% Serum, 2hours following 0.25% serum starvation, 36hours) treated HeLa and 293Twhole cell lysate, respectively, onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% Milk/TBST for at least 1 hour. Membranes were probed with a rabbit polyclonal antibody recognizing c-Fos at a dilution of 1:500 overnight at 4°C on a rocking platform. Membranes were washed in TBS-0.1%Tween 20 and probed with a goat anti-rabbit-HRP secondary antibody (Product # 31460) at a dilution of 1:20,000 for at least one hour. Membranes were washed and chemiluminescent detection performed using Pierce Super Signal West Pico (Product # 34077).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis was performed on whole cell extracts (30 µg lysate) of HEL 92.1.7 (Lane 1), Jurkat (Lane 2), HeLa (Lane 3), HeLa treated for 20 minutes with 200 mM of TPA (Lane 4), K562 (lane5), KARPAS-299 (Lane 6) and HT-29 (lane 7). The blots were probed with Anti-c-Fos Rabbit Polyclonal Antibody (Product # PA1-830, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 29 kDa band corresponding to c-Fos was observed across cell lines tested, A 40 kDa isoform was observed in TPA treated lysate. 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)
- Main image
- Experimental details
- Immunofluorescent analysis of c-FOS (green) in untreated HeLa cells. Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature. Cells were then blocked with 5% normal goat serum (Product # 31873) for 15 minutes at room temperature. Cells were probed with a rabbit polyclonal antibody recognizing c-FOS (Product # PA1-830), at a dilution of 1:500 for at least 1 hour at room temperature. Cells were washed with PBS and incubated with DyLight 488 goat-anti-rabbit secondary antibody at a dilution of 1:400 for 30 minutes at room temperature. Nuclei were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan at 20X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow cytometry analysis of c-FOS was done on HeLa cells treated with PMA (200nM, 20 minutes). Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with c-FOS Rabbit Polyclonal Antibody (PA1830, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 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.
