ALDH1L1 antibody (ab56777) used in immunohistochemistry at 3ug/ml on formalin fixed and paraffin embedded human kidney.
ICC/IF image of ab56777 stained HeLa 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 (ab56777, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.
Overlay histogram showing HepG2 cells stained with ab56777 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab56777, 0.5µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was Mouse IgG3 [MG3-35] (ab18394, 1µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
ALDH1L1 was immunoprecipitated using 0.5mg Mouse Liver whole tissue extract, 5µg of Mouse monoclonal to ALDH1L1 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 Liver whole tissue 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 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab56777.Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.Band: 99kDa: ALDH1L1; non specific - 60kDa: We are unsure as to the identity of this extra band.