The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 68 kDa (predicted molecular weight: 49 kDa).
Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Recruited and tyrosine phosphorylated by several receptor systems, for example the T-cell, leptin and insulin receptors. Once phosphorylated, functions as an adapter protein in signal transduction cascades by binding to SH2 and SH3 domain-containing proteins. Role in G2-M progression in the cell cycle. Represses CBP-dependent transcriptional activation apparently by competing with other nuclear factors for binding to CBP. Also acts as a putative regulator of mRNA stability and/or translation rates and mediates mRNA nuclear export. Isoform 3, which is expressed in growth-arrested cells only, inhibits S phase.
Ubiquitously expressed in all tissue examined. Isoform 1 is expressed at lower levels in brain, skeletal muscle, and liver whereas isoform 3 is intensified in skeletal muscle and in liver.
Belongs to the KHDRBS family. Contains 1 KH domain.
Isoform 3 is only expressed in growth-arrested cells.
The KH domain is required for binding to RNA. The Pro-rich domains are flanked by Arg/Gly-rich motifs which can be asymmetric dimethylated on arginine residues to give the DMA/Gly-rich regions. Selective methylation on these motifs can modulate protein-protein interactions.
Tyrosine phosphorylated by several non-receptor tyrosine kinases, for example LCK, FYN and JAK3. Negatively correlates with ability to bind RNA but required for many interactions with proteins. Acetylated. Positively correlates with ability to bind RNA. Arginine methylation is required for nuclear localization. Also can affect interaction with other proteins. Inhibits interaction with Src-like SH3 domains, but not interaction with WW domains of WBP4/FBP21 AND FNBP4/FBP30. Arg-291, Arg-331 and Arg-346 are found to be also dimethylated, probably to asymmetric dimethylarginine.
ICC/IF image of ab26803 stained human HEK 293 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab26803, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, HepG2 and MCF7 cells.
Western blot - Anti-SAM68 antibody (ab26803)This image is courtesy of an anonymous Abreview
Anti-SAM68 antibody (ab26803) at 1/600 dilution (for 20 hours at 4°C in blocking buffer) + Whole cell lysate of human fibroblasts at 20 µg
Secondary An IRDye®800CW Donkey anti-rabbit IgG polyclonal at 1/3000 dilution
IHC image of ab26803 staining in human skin 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 ab26803, 1µg/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.
Palmer SJ et al. GTF2IRD2 from the Williams-Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members. J Cell Sci125:5040-50 (2012).
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