ab45231 gave a positive result in the following whole cell lysates: Hela (data not shown), Jurkat, A431, HEK293 (data not shown), MCF7, U20S (data not shown).
IHC-P: human colon adenocarcinoma FFPE tissue sections
This antibody gave a positive result when used in the following formaldehyde fixed cell lines: HepG2.
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 36 kDa (predicted molecular weight: 42 kDa).
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use a concentration of 5 µg/ml.
Required for the control of apoptosis during postnatal growth. Essential for proteolytic processing of an antiapoptotic form of OPA1 which prevents the release of mitochondrial cytochrome c in response to intrinsic apoptoptic signals (By similarity). Promotes changes in mitochondria morphology regulated by phosphorylation of P-beta domain.
Belongs to the peptidase S54 family.
P-beta is proteolytically processed (beta-cleavage) in a PARL-dependent manner. The cleavage is inhibited when residues Ser-65, Thr-69 and Ser-70 are all phosphorylated.
Mitochondrion inner membrane and Nucleus. Translocated into the nucleus by an unknown mechanism.
ICC/IF image of ab45231 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 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 ab45231 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 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.
Western blot - PARL antibody (ab45231)
All lanes : Anti-PARL antibody (ab45231) at 1 µg/ml
Lane 1 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate Lane 2 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 3 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution
PARL is a mitochondrial protein which is constitutively cleaved, leaving a protein of 36.5 kDa (Sík A et al, J Biol Chem, 2004 Apr 9;279(15):15323-9 (PMID 14732705)). ab45231 detects a band at 36 kDa (cleaved form). However, Abcam's data suggests that ab45231 does not detect the full PARL protein (42 kDa).
IHC image of PARL staining in human colon adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab45231, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.