Anti-RIP 抗体 [EPR24883-85] (ab300617)

Anti-RIPK1 antibody [EPR24883-85] (ab300617) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab300617 was shown to bind specifically to RIPK1. A band was observed at 75 kDa in wild-type THP-1 cell lysates with no signal observed at this size in RIPK1 knockout cell line ab276121 (knockout cell lysate ab284221). To generate this image, wild-type and RIPK1 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween$®$ 20 (TBS-T) 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

False colour image of Western blot: Anti-RIP antibody [EPR24883-85] (ab300617) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

Blocking / Diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

WB performed under reducing conditions.

ab300617 was shown to bind specifically to RIP. A band was observed at 75 kDa in wild-type HAP1 cell lysates with no signal observed at this size in RIP knockout cell line. To generate this image, wild-type and RIP knockout HAP1 cell lysates were analyzed. First, samples were run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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.

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

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

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

Exposure time:

Lane 1: 37 seconds; lane 2: 180 seconds.

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma tissue labelling RIP with ab300617 at 1/100 dilution (5.11 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Cytoplasmic staining on human cervical carcinoma was observed. The section was incubated with ab300617 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 was performed with Tris-EDTA buffer (pH 9.0 epitope retrieval solution 2) for 20 mins.

Immunohistochemical analysis of paraffin-embedded Wild-type and RIPK1 knockout HAP1 cells labelling RIP with ab300617 at 1/100 dilution (5.11 µg/mL) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on Wild-type HAP1 cell pellet (A), and no staining on RIPK1 knockout HAP1 cell pellet (B) were observed. The section was incubated with ab300617 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 was performed with Tris-EDTA buffer (pH 9.0 epitope retrieval solution 2) for 20 mins.

RIP was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab300617 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300617 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 µg (Input)

Lane 2: ab300617 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300617 in HeLa whole cell lysate

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

Exposure time: 41 seconds

RIP was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab300617 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab300617 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 µg (Input)

Lane 2: ab300617 IP in NIH/3T3 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300617 in NIH/3T3 whole cell lysate

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

Exposure time: 3 minutes