This antibody gave a positive signal in nuclear extracts from the following cell lines: HepG2; MCF7.
IHC-P (FFPE): Human Placenta (Normal) tissue.
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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 1 µg/ml. Detects a band of approximately 79 kDa (predicted molecular weight: 79 kDa).
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Transcriptional repressor involved in developmental processes. Probably plays a role in limb pattern formation.
Involvement in disease
Defects in TBX3 are the cause of ulnar-mammary syndrome (UMS) [MIM:181450]. UMS is characterized by ulnar ray defects, obesity, hypogenitalism, delayed puberty, hypoplasia of nipples and apocrine glands.
ICC/IF image of ab99302 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 ab99302 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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. This antibody also gave a positive result in formaldehyde fixed (4%, 10min) HepG2 cells at 5ug/ml.
IHC image of Tbx3 staining in Human Placenta 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 ab99302, 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.
Western blot - Anti-Tbx3 antibody (ab99302)
All lanes : Anti-Tbx3 antibody (ab99302) at 1 µg/ml
Lane 1 : HepG2 (Human hepatocellular liver carcinoma cell line) Nuclear Lysate Lane 2 :MCF7 nuclear extract lysate (ab14860)
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 79 kDa Observed band size: 79 kDa Additional bands at: 22 kDa, 77 kDa (possible isoform). We are unsure as to the identity of these extra bands.
Exposure time: 3 minutes
Ab99302 should recognise all three isoforms of Tbx3. We are probably observing Isoform I and Isoform II on this Western Blot image. The molecular weights of Isoform I and Isoform II are 77- and 79-kDa respectively.
Immunocytochemistry/ Immunofluorescence - Anti-Tbx3 antibody (ab99302)This image is courtesy of an Abreview by Zoe Burke.
ab99302 staining Tbx3 in the AR42J-B13 rat pancreatic tumor cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton/PBS and blocked with blocking buffer for 60 minutes at room temperature. Samples were incubated with primary antibody (1/100) for 18 hours at 4°C. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG polyclonal was used as the secondary antibody at a dilution of 1/5000.