Immunofluorescence analysis of Histone H3.3 was performed using 70% confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Histone H3.3 Rabbit Polyclonal Antibody (PA529602) at 2µg/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 (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 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.
|Tested species reactivity||Human, Mouse, Rat|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant protein fragment corresponding to a region within amino acids 1 and 136 of Human Histone H3.3|
|Purification||Antigen affinity chromatography|
|Storage buffer||PBS, pH 7, with 20% glycerol|
|Storage Conditions||-20° C, Avoid Freeze/Thaw Cycles|
|Tested Applications||Dilution *|
|ChIP assay (ChIP)||1.5 µg/10^6 cells|
|Immunocytochemistry (ICC)||2 µg/ml|
|Immunofluorescence (IF)||2 µg/ml|
|Immunohistochemistry (Paraffin) (IHC (P))||1:100-1:1000|
|Western Blot (WB)||1:5,000|
* 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.
PA5-29602 targets Histone H3.3 in WB and IHC-P applications and shows reactivity with Human, mouse and rat samples.
The PA5-29602 immunogen is recombinant protein fragment corresponding to a region within amino acids 1 and 136 of Human Histone H3.3.
Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene contains introns and its mRNA is polyadenylated, unlike most histone genes. The protein encoded is a replication-independent member of the histone H3 family.
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