This antibody gave a positive signal in the following lysates: Human Brain Tissue; Human Testis Tissue; Mouse Hippocampus Tissue; Mouse Testis Tissue; HeLa Whole Cell; Jurkat Whole Cell. It also gave a positive signal in FFPE human hippocampus tissue sections.
This antibody gave a positive result when used in the following formaldehyde fixed cell lines: PC12
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ICC/IF image of ab87498 stained PC12 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 ab87498 at 10µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-Cpt1c antibody (ab87498)
All lanes : Anti-Cpt1c antibody (ab87498) at 1 µg/ml
Lane 1 : Human brain tissue lysate - total protein (ab29466) Lane 2 : Human testis tissue lysate - total protein (ab30257) Lane 3 : Hippocampus (Mouse) Tissue Lysate Lane 4 : Testis (Mouse) Tissue Lysate Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 6 : Jurkat (Human T cell lymphoblast-like cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution
ICC/IF image of ab87498 stained HeLa cells. The cells were 4% PFA 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 (ab87498, 5µg/ml) 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.
IHC image of ab87498 staining in human hippocampus 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 ab87498, 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.
Cpt1c was immunoprecipitated using 0.5mg Mouse Testis tissue lysate, 5µg of Rabbit polyclonal to Cpt1c and 50µl of protein G magnetic beads (+). No antibody was added to the control (-). The antibody was incubated under agitation with Protein G beads for 10min, Mouse Testis tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation. Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab87498. Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697). Band: 99kDa, non specific bands - 50 and 55kDa: We are unsure as to the identity of this extra band; Cpt1c.