The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 2.5 - 5 µg/ml. Detects a band of approximately 13 kDa (predicted molecular weight: 10 kDa).
RelevanceApoptosis plays a major role in normal organism development, tissue homeostasis, and removal of damaged cells. Hrk, a pro-apoptotic member of the Bcl-2 homology domain-3 (BH3) group of the Bcl-2 family of proteins, was identified as a novel protein induced during programmed neuronal death. It lacks significant homology to other Bcl-2 family members except for an 8-amino acid region that is similar to the BH3 motif of Bik. Hrk regulates apoptosis through interaction with the anti-apoptotic proteins Bcl-2 and Bcl-XL via this domain. It does not interact with the pro-apoptotic proteins Bax, Bak, or Bcl-XS. Hrk localizes to mitochondrial membranes in a pattern similar to that previously reported for Bcl-2 and Bcl-XL. Despite its predicted molecular weight, Hrk often migrates at 12-15 kDa.
ab45419 (2ug/ml) staining Hrk in human testis using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining of Hrk in cytoplasm of the germinal epithelium. Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 using a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
ICC/IF image of ab45419 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab45419, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
References for Anti-Hrk antibody (ab45419)
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