Recombinant Anti-TLS/FUS antibody [EPR5812] (ab124923)

We are unsure about the nature of the extra bands.

ab124923 was shown to react with FUS in wild-type HeLa cells in Western blot with loss of signal observed in a FUS knockout cell line. Wild-type HeLa and FUS knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab124923 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 1/5000 before imaging. These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

Lanes 1- 2: Merged signal (red and green). Green - ab124923 observed at 75 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab124923 was shown to react with TLS/FUS in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266587 (knockout cell lysate ab257100) was used. Wild-type HEK-293T and FUS knockout HEK-293T 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. ab124923 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.

Immunocytochemistry/ Immunofluorescence analysis of K-562 (human chronic myelogenous leukemia lymphoblast) cells labeling TLS/FUS with ab124923 at 1/250 (8.3 μg/mL). Cells were fixed with 4% Paraformaldehyde and permeabilised 0.1% TritonX-100. ab150077, AlexaFluor^®488 Goat anti-Rabbit secondary at 1/1000 (2 μg/mL) was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

Confocal image showing nuclear staining in K-562 cells.

Lanes 1 to 4: Merged signal (red and green). Green - ab124923 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab124923 was shown to specifically react with TLS/FUS when TLS/FUS knockout samples were used. Wild-type and TLS/FUS knockout samples were subjected to SDS-PAGE. ab124923 and ab28245 (loading control to GAPDH) were both diluted at 1/1000 and 1/10 000 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 1/10 000 dilution for 1 h at room temperature before imaging.

ab124923, at 1/50 dilution, staining TLS/FUS in formalin-fixed, paraffin-embedded Human kidney tissue, by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.