Recombinant Anti-USP10 antibody [EPR4261] (ab109219)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: USP10 knockout HAP1 cell lysate (20 µg)
Lane 3: A549 cell lysate (20 µg)
Lane 4: A375 cell lysate (20 µg)
Lanes 1 to 4: Merged signal (red and green). Green - ab109219 observed at 115 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab109219 was shown to recognize USP10 when USP10 knockout samples were used, along with additional cross-reactive bands. Wild-type and USP10 knockout samples were subjected to SDS-PAGE. ab109219 and ab8245 (loading control to GAPDH) were both diluted at 1/1000 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

ab109219, at a 1/250 dilution, staining USP10 in paraffin embedded Human colon tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ICC/IF image of ab109219 stained MCF-7 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 ab109219 at 1/50 dilution 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.

USP10 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg with 109219 at 1/30 dilution (2µg) . VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg

Lane 2: ab109219 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab109219 in HeLa whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Fresh lysate should be used to minimize protein degradation.