Anti-EBP50/NHERF-1 抗体 [EPR5562] (ab109430)

False colour image of Western blot: Anti-EBP50/NHERF-1 antibody [EPR5562] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab109430 was shown to bind specifically to EBP50/NHERF-1. A band was observed at 46 kDa in wild-type HeLa cell lysates with no signal observed at this size in SLC9A3R1 knockout cell line ab264914 (knockout cell lysate ab257280). To generate this image, wild-type and SLC9A3R1 knockout HeLa cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Lanes 1- 2: Merged signal (red and green). Green - ab109430 observed at 48 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab109430 was shown to react with EBP50/NHERF-1 in wild-type HCT116 cells in western blot. Loss of signal was observed when knockout cell line ab266876 (knockout cell lysate ab257281) was used. Wild-type HCT116 and SLC9A3R1 knockout HCT116 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab109430 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of EBP50/NHERF-1 in paraffin-embedded Human kidney tissue using ab109430 at 1/100 dilution.

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

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: EBP50/NHERF-1 knockout HAP1 cell lysate (20 µg)
Lane 3: Jurkat cell lysate (20 µg)
Lane 4: HepG2 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab109430 observed at 48 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab109430 was shown to specifically recognize EBP50/NHERF-1 in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when EBP50/NHERF-1 knockout samples were usexamined. Wild-type and EBP50 knockout samples were subjected to SDS-PAGE. ab109430 and ab18058 (loading control to Vinculin) were diluted at 1/500 and 1/10000 respectively and incubated overnight at 4°C. Blots were developed withGoat 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 hour at room temperature before imaging.