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Purified Mouse Anti-NF-κB p65
Purified Mouse Anti-NF-κB p65

Western blot analysis of NF-κB p65 on a Jurkat lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of NF-κB p65 antibody.

Purified Mouse Anti-NF-κB p65

Immunofluorescent staining of A549 (ATCC CCL-185) cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were either mock treated (media, left) or exposed to TNF  (20ng/ml, right) for 15 minutes.  After treatment cells were stained using the Triton™ X-100 perm protocol and the anti-NF-κB antibody.  The second step reagent was Alexa-Fluor® 488 goat anti-mouse IgG (Invitrogen).  The image was taken on a BD Pathway™ 855 Bioimager with a 20x objective.  This antibody also stains U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells and can be used with either perm protocol (see Recommended Assay Procedure).

Western blot analysis of NF-κB p65 on a Jurkat lysate. Lane 1: 1:250, lane 2: 1:500, lane 3: 1:1000 dilution of NF-κB p65 antibody.

Immunofluorescent staining of A549 (ATCC CCL-185) cells.  Cells were seeded in a 96 well imaging plate (Cat. No. 353219) at ~ 10 000 cells per well.  After overnight incubation, cells were either mock treated (media, left) or exposed to TNF  (20ng/ml, right) for 15 minutes.  After treatment cells were stained using the Triton™ X-100 perm protocol and the anti-NF-κB antibody.  The second step reagent was Alexa-Fluor® 488 goat anti-mouse IgG (Invitrogen).  The image was taken on a BD Pathway™ 855 Bioimager with a 20x objective.  This antibody also stains U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells and can be used with either perm protocol (see Recommended Assay Procedure).

製品詳細
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BD Transduction Laboratories™
Human (QC Testing), Rat, Dog, Frog, Rabbit (Tested in Development)
Mouse IgG1
Human NF-κB aa. 136-224
Western blot (Routinely Tested), Bioimaging (Tested During Development)
65 kDa
250 µg/ml
AB_398186
Aqueous buffered solution containing BSA, glycerol, and ≤0.09% sodium azide.
RUO


推奨アッセイ手順

Bioimaging

1. Seed the cells in appropriate culture medium at ~10,000 cells per well in a BD Falcon™ 96-well Imaging Plate (Cat. No. 353219) and culture overnight.

2. Remove the culture medium from the wells, and fix the cells by adding 100 μl of BD Cytofix™ Fixation Buffer (Cat. No. 554655) to each well.  Incubate for 10 minutes at room temperature (RT).

3. Remove the fixative from the wells, and permeabilize the cells using either BD Perm Buffer III, 90% methanol, or Triton™ X-100:

a. Add 100 μl of -20°C 90% methanol or Perm Buffer III (Cat. No. 558050) to each well and incubate for 5 minutes at RT.

     OR

b. Add 100 μl of 0.1% Triton™ X-100 to each well and incubate for 5 minutes at RT.

4. Remove the permeabilization buffer, and wash the wells twice with 100 μl of 1× PBS.

5. Remove the PBS, and block the cells by adding 100 μl of BD Pharmingen™ Stain Buffer (FBS) (Cat. No. 554656) to each well. Incubate for 30 minutes at RT.

6. Remove the blocking buffer and add 50 μl of the optimally titrated primary antibody (diluted in Stain Buffer) to each well, and incubate for 1 hour at RT.

7. Remove the primary antibody, and wash the wells three times with 100 μl of 1× PBS.

8. Remove the PBS, and add the second step reagent at its optimally titrated concentration in 50 μl to each well, and incubate in the dark for 1 hour at RT.

9. Remove the second step reagent, and wash the wells three times with 100 μl of 1× PBS.

10. Remove the PBS, and counter-stain the nuclei by adding 200 μl per well of 2 μg/ml Hoechst 33342 (e.g., Sigma-Aldrich Cat. No. B2261) in 1× PBS to each well at least 15 minutes before imaging.

11. View and analyze the cells on an appropriate imaging instrument.

Bioimaging:  For more detailed information please refer to http://www.bdbiosciences.com/support/resources/protocols/ceritifed_reagents.jsp

Western blot:  For more detailed information please refer to http://www.bdbiosciences.com/pharmingen/protocols/Western_Blotting.shtml

製品通知

  1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
  2. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
  3. This antibody has been developed and certified for the bioimaging application. However, a routine bioimaging test is not performed on every lot. Researchers are encouraged to titrate the reagent for optimal performance.
  4. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
  5. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
  6. Triton is a trademark of the Dow Chemical Company.
610868 Rev. 3
抗体の詳細
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20/NF-kB/p65

NF-κB is a ubiquitously expressed transcription factor that regulates many cytokine and Ig genes. It is involved in immune, inflammatory, viral, and acute phase responses. The most studied NF-κB complex consists of the p50 and p65 subunits, both containing a 300 amino acid region with homology to the Rel proto-oncogene product. The p50 subunit binds DNA, whereas the p65 subunit is responsible for the interaction of NF-κB with its inhibitor, IκB. In most cell types, the p50/p65 heterodimer is located within the cytoplasm complexed to IκB. This complex prevents nuclear translocation and activity of NF-κB. In response to stimuli such as cytokines, LPS, and viral infections, IκB is phosphorylated at critical residues. This phosphorylation induces dissociation of the IκB/NF-κB complex, allowing the free heterodimeric NF-κB to form a heterotetramer that translocates to the nucleus. In the nucleus, it binds to the κB site within promoters and enhancers and functions as a transcriptional activator.

610868 Rev. 3
フォーマットの詳細
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Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
610868 Rev.3
引用&参考文献
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開発者向け参考資料 (5)

  1. Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988; 53(2):211-217. (Biology). 参考文献を見る
  2. Kieran M, Blank V, Logeat F, et al. The DNA binding subunit of NF-kappa B is identical to factor KBF1 and homologous to the rel oncogene product. Cell. 1990; 62(5):1007-1018. (Biology). 参考文献を見る
  3. Nishibe T, Parry G, Ishida A, et al. Oncostatin M promotes biphasic tissue factor expression in smooth muscle cells: evidence for Erk-1/2 activation. Blood. 2001; 97(3):692-699. (Clone-specific: Western blot). 参考文献を見る
  4. Pimentel-Muinos FX, Seed B. Regulated commitment of TNF receptor signaling: a molecular switch for death or activation. Immunity. 1999; 11(6):783-793. (Clone-specific: Western blot). 参考文献を見る
  5. Shirakawa F, Mizel SB. In vitro activation and nuclear translocation of NF-kappa B catalyzed by cyclic AMP-dependent protein kinase and protein kinase C. Mol Cell Biol. 1989; 9(6):2424-2430. (Biology). 参考文献を見る
すべて表示する (5) 表示項目を減らす
610868 Rev. 3

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