Immunofluorescence analysis of NF-kB p65 was done on 70% 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 5% BSA for 1 hour at room temperature. The cells were labeled with NF-kB p65 Rabbit Polyclonal Antibody (PA1186) at 1ug/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) 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 (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing cytoplasmic and nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse|
|Host / Isotype||Rabbit / IgG|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS with 30% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1-5 µg x 10^6 cells|
|Flow Cytometry (Flow)||3-5 µg/10^6 cells|
|Immunocytochemistry (ICC)||1-2 µg/ml|
|Immunofluorescence (IF)||1-2 µg/ml|
|Immunoprecipitation (IP)||3 µg|
|Western Blot (WB)||1-3 µg/ml|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
PA1-186 has been successfully used in WB using StartingBlock T20 (TBS) Blocking Buffer (Product # 37543). More non-specific bands are observed when using 5% BSA for blocking.
By WB, PA1-186 detects a predominant band at 65kD. Loading increasing amounts of cell lysate may yield more intense nonspecific bands at ~120kD and/or ~50kD.
PA1-186 has been succesfully used for IP of the NF-kB p65 subunit and co-IP of the NF-kB p50 subunit.
NF-kB (nuclear factor kB) regulates the expression of a large number of genes that play critical roles in apoptosis, viral replication, tumorigenesis, various autoimmune diseases, and inflammation. The active nuclear form of the NF-kB transcription factor complex is composed of two DNA binding subunits, NF-kB p65 and p50, both of which share extensive N-terminal sequence homology with the v-rel oncogene product. N-terminal regions of p50 and p65 are critical for DNA binding and help determine the DNA-binding specificities of p50 and p65.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.