Recombinant Anti-TRIM25/EFP antibody [EPR7315] (ab167154)

Lanes 1 - 4: Merged signal (red and green). Green - ab167154 observed at 71 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab167154 was shown to specifically react with TRIM25/EFP in wild-type HAP1 cells as signal was lost in TRIM25/EFP knockout cells. Wild-type and TRIM25/EFP knockout samples were subjected to SDS-PAGE. Ab167154 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/10000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Blocking and diluting buffer: 5% NFDM/TBST

ab167154 staining TRIM25/EFP in human pancreas tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and antigen retrieval was by heat mediation in a EDTA buffer. Samples were incubated with primary antibody at a dilution of 1/100. A goat anti-rabbit IgG H&L (HRP) ab97051 was used as the secondary antibody at a dilution of 1/500.

ab167154 staining TRIM25/EFP in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. ab195889 was used as a tubulin counterstain at a dilution of 1/200 and DAPI was used as a nuclear counterstain.

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling TRIM25/EFP with purified ab167154 at 1/90 dilution(10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

Blocking and diluting buffer: 5% NFDM /TBST 

ab167154 immunoprecipitating TRIM25/EFP. 10µg of cell lysate was incubated with primary antibody at a dilution of 1/40 and VeriBlot for IP Detection Reagent (HRP) (ab131366) at a dilution of 1/1000.

Lane 1: HeLa (human cervix adenocarcinoma) whole cell lysate (10ug)
Lane 2: HeLa (human cervix adenocarcinoma) whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab167154 in HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate.

Blocking and diluting buffer: 5% NFDM/TBST

Immunohistochemical analysis of paraffin-embedded Human breast tissue labeling TRIM25/EFP with ab167154 at 1/100 dilution.

Immunofluorescent analysis of HeLa cells labeling TRIM25/EFP with ab167154 at 1/100 dilution.