Recombinant Anti-Sigma1-receptor antibody [EPR23266-69] (ab253192)

ab253192 was shown to react with SIGMAR1 in wild-type HEK293T in Western blot with loss of signal observed in SIGMAR1 knockout cell line ab266619 (SIGMAR1 knockout cell lysate ab258666). Wild-type HEK293T and HeLa cell lysates and the corresponding SIGMAR1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab253192 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 0.2μg/mL 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 - 4: Merged signal (red and green). Green - ab253192 observed at 24 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55 kDa.

ab253192 was shown to react with Sigma1-receptor in wild-type HEK-293T cells in Western blot with loss of signal observed in SIGMAR1 knockout cell line ab266619 (SIGMAR1 knockout cell lysate ab258666). Wild-type HEK-293T and SIGMAR1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab253192 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labelling Sigma1-receptor with ab253192 at 1/600 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling Sigma1-receptor with ab253192 at 1/500 dilution (1.23 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat liver (PMID: 8954936). The section was incubated with ab253192 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Exposure time: 3.25 seconds

Blocking and diluting buffer and concentration: 5% NFDM/TBST   

Exposure time: 3 minutes

Blocking and diluting buffer and concentration: 5% NFDM/TBST 

Exposure time: 3 minutes

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling Sigma1-receptor with ab253192 at 1/500 dilution (1.23 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human liver (PMID: 8954936). The section was incubated with ab253192 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Rat spinal cord tissue labeling Sigma1-receptor with ab253192 at 1/500 dilution (1.23 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat spinal cord (PMID: 23314020). The section was incubated with ab253192 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human spinal cord tissue labeling Sigma1-receptor with ab253192 at 1/500 dilution (1.23 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human spinal cord (PMID: 23314020). The section was incubated with ab253192 for 30 mins at room temperature.The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins