Recombinant Anti-TMS1/ASC antibody [EPR23978-28] (ab283684)

Blocking and diluting buffer and concentration: 3% NFDM/TBST

Negative control: HeLa (PMID:11103776;10567338).

Lanes 1-4: Merged signal (red and green). Green - ab283684 observed at 24 kDa. Red - loading control ab8245 observed at 36 kDa.

Ab283684 Anti-TMS1/ASC antibody [EPR23978-28] was shown to specifically react with TMS1/ASC in wild-type HAP1 cells. Loss of signal was observed when knockout cell line (knockout cell lysate) was used. Wild-type and TMS1/ASC knockout samples were subjected to SDS-PAGE. ab283684 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 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. 

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

ASC has four isoforms. 24-kDa ASC, 17-kDa ASC-b and 12-kDa ASC-C are observed. The molecular weight observed is consistent with what has been described in the literature (PMID:20482797).

Exposure time: 81 seconds

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labelling TMS1/ASC with ab283684 at 1/2000 (0.247 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human tonsil. The section was incubated with ab283684 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling TMS1/ASC with ab283684 at 1/2000 (0.247 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human colon. The section was incubated with ab283684 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 LeicaDS9800 (Bond™ Polymer Refine Detection) was used.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunohistochemical analysis of paraffin-embedded Human colon cancer tissue labelling TMS1/ASC with ab283684 at 1/2000 (0.247 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on human colon cancer (PMID: 14643031). The section was incubated with ab283684 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 LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling TMS1/ASC with ab283684 at 1/100 (4.94 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing cytoplasmic taining in THP-1 cell line. Negative control: HeLa (PMID: 11103776). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell, Left) / THP-1 (Human monocytic leukemia monocyte, Right) cells labelling TMS1/ASC with ab283684 at 1/5000 dilution (0.01ug) (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, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: HeLa (PMID: 11103776).

TMS1/ASC was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate 20 ug with ab283684 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283684 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (human monocytic leukemia monocyte) whole cell lysate 20 ug

Lane 2: ab283684 IP in THP-1 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab283684 in THP-1 whole cell lysate

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

Exposure time: 10 seconds