![TLE3 was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to TLE3 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, HeLa whole cell extract 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab94972.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 83kDa; TLE3](http://www.bioprodhub.com/system/product_images/ab_products/2/sub_5/11534_ab94972-203453-IPVI001ab9497220m.jpg)
TLE3 was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to TLE3 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, HeLa whole cell extract 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab94972.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 83kDa; TLE3
All lanes : Anti-TLE3 antibody (ab94972) at 1 µg/mlLane 1 : Breast (Human) Tissue Lysate - normal tissue (ab30090)Lane 2 : MCF7 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 3 : MDA-MB-231 (Human breast adenocarcinoma cell line) Whole Cell Lysate Lane 4 : Lung (Human) Tissue Lysate Lane 5 : A549 (Human lung adenocarcinoma epithelial cell line) Whole Cell Lysate Lane 6 : A431 (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 7 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate Lysates/proteins at 10 µg per lane.SecondaryGoat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.
ICC/IF image of ab94972 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 (ab94972, 1µg/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. This antibody also gave a positive result in 4% PFA fixed (10 min) Hek293, HepG2 and MCF7 cells at 1µg/ml.