The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
1/1000. Detects a band of approximately 29 kDa (predicted molecular weight: 15 kDa).
ID (inhibitor of DNA binding) HLH proteins lack a basic DNA-binding domain but are able to form heterodimers with other HLH proteins, thereby inhibiting DNA binding. ID-2 may be an inhibitor of tissue-specific gene expression.
Highly expressed in early fetal tissues, including those of the central nervous system.
Contains 1 basic helix-loop-helix (bHLH) domain.
Found in most early fetal tissues but not in the corresponding mature tissues.
IHC image of ID2 staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab85990, 1/1000, 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.
Western blot - Anti-ID2 antibody (ab85990)
All lanes : Anti-ID2 antibody (ab85990) at 1/1000 dilution
Lane 1 : Uninduced (negative control) culture of E.coli Lane 2 : Induced culture of E.coli
Secondary All lanes : anti-Rabbit IgG HRP conjugated at 1/1000 dilution
ICC/IF image of ab85990 stained NIH-3T3 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 (ab85990, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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.
Ladle BH et al. De novo DNA methylation by DNA methyltransferase 3a controls early effector CD8+ T-cell fate decisions following activation. Proc Natl Acad Sci U S A113:10631-6 (2016).
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