Immunofluorescent analysis of KAP1 (green) showing staining in the nucleus of NIH-3T3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a KAP1 polyclonal antibody (Product # PA1-852) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4°C in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Actin was stained using Alexa Fluor 554 (red) and nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.
|Tested species reactivity||Human, Mouse, Rat|
|Published species reactivity||Mouse|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant human KAP-1.|
|Purification||Ammonium sulfate precipitation|
|Contains||0.05% sodium azide|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|Immunohistochemistry (Paraffin) (IHC (P))||1:50-1:500|
|Western Blot (WB)||1:500|
* 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.
|Immunocytochemistry (ICC)||See 1 publications below|
PA1-852 detects KAP-1 (Kruppel-associated box-associated protein 1) in human, mouse and rat samples.
PA1-852 has been successfully used in Western blot, ICC/IF and IHC (P) procedures. By Western blot, this antibody detects an ~87 kDa protein representing recombinant human KAP-1. This antibody also detects a smaller protein that is believed to be KAP-1 degradation product. In immunofluorescent applications, this antibody primarily reacts with mouse or rat and shows non-specific staining in HeLa cells.
The PA1-852 antigen is a recombinant human KAP-1.
The KRAB (Kruppel-associated box) domain is minimally about 45 amino acids in length and is a transcriptional repression domain found in numerous transcription factors. There have been identified over 220 KRAB-zinc finger protein (KRAB-ZFP) genes in the human genome. These proteins functionally repress transcription via specific interactions with KAP-1 (KRAB-associated protein 1). KAP-1 is an 835 amino acid polypeptide that contains a RING finger, B boxes, and a PHD finger. KAP-1 has been shown to form complexes with KRAB-domain transcription factors and increase the efficiency with which they mediate repression. KAP-1 has also been shown to directly interact with HP1 (heterochromatin protein 1) and KRAZ1 (Kruppel-associated box-containing zinc finger protein 1). KAP-1 directly targets KRAZ1 to the foci of centromeric heterochromatin containing HP1alpha, thus helping to regulate transcriptional repression. Recent studies have shown that KAP-1 mutants with the ability to bind KRAB but unable to bind HP1 leads to random distribution of KRAZ1 and strong transcriptional activation.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Targeting of Krüppel-associated box-containing zinc finger proteins to centromeric heterochromatin. Implication for the gene silencing mechanisms.
PA1-852 was used in immunocytochemistry to investigate the relationship between KRAB-ZFPs and centromeric heterochromatin and their interaction with KAP-1
|Matsuda E,Agata Y,Sugai M,Katakai T,Gonda H,Shimizu A||The Journal of biological chemistry (276:14222)||2001|