Anti-MyD88 antibody [EPR21824]

4 product images

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  Western blot - Anti-MyD88 antibody [EPR21824] (ab219413)

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  ab219413 was shown to specifically react with MyD88 in wild-type HAP1 cells as signal was lost in MyD88 knockout cells. Wild-type and MyD88 knockout samples were subjected to SDS-PAGE. ab219413 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique

  All lanes: Western blot - Anti-MyD88 antibody [EPR21824] (ab219413) at 1/100000 dilution

  Lane 1: Wild-type HAP1 whole cell lysate at 20 µg

  Lane 2: MyD88 knockout HAP1 whole cell lysate at 20 µg

  Lane 3: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 33 kDa

  Exposure time: 3min

  Blocking/Dilution buffer: 5% NFDM/TBST.
  

  ab219413 was shown to specifically react with MyD88 in wild-type HAP1 cells as signal was lost in MyD88 knockout cells. Wild-type and MyD88 knockout samples were subjected to SDS-PAGE. ab219413 and Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging. The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab219413).

• 

  Immunoprecipitation - Anti-MyD88 antibody [EPR21824] (ab219413)

  MyD88 was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate with ab219413 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab219413 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/5000 dilution.
  

  
  Lane 1: RAW 264.7 whole cell lysate 10 μg (Input).
  Lane 2: ab219413 IP in RAW 264.7 whole cell lysate (+).
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab219413 in RAW 264.7 whole cell lysate (-).
  Blocking/Dilution buffer: 5% NFDM/TBST.
  Exposure time: 3 minutes.

  All lanes: Immunoprecipitation - Anti-MyD88 antibody [EPR21824] (ab219413)

  Predicted band size: 33 kDa

• 

  Western blot - Anti-MyD88 antibody [EPR21824] (ab219413)

  Blocking/Dilution buffer: 5% NFDM/TBST

  All lanes: Western blot - Anti-MyD88 antibody [EPR21824] (ab219413) at 1/1000 dilution

  Lane 1: Rat liver lysate at 20 µg

  Lane 2: RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

  Lane 3: A20 (Mouse reticulum sarcoma B lymphocyte) cell lysate at 20 µg

  Lane 4: Mouse liver lysate at 20 µg

  Lane 5: Mouse lung lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Predicted band size: 33 kDa

  Exposure time: 3min

• 

  Western blot - Anti-MyD88 antibody [EPR21824] (ab219413)

  False colour image of Western blot: Anti-MyD88 antibody [EPR21824] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab219413 was shown to bind specifically to MyD88. A band was observed at 35 kDa in wild-type A549 cell lysates with no signal observed at this size in MYD88 knockout cell line Human MYD88 knockout A549 cell line ab286715 (knockout cell lysate ab290793). To generate this image, wild-type and MYD88 knockout A549 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.

  All lanes: Western blot - Anti-MyD88 antibody [EPR21824] (ab219413) at 1/1000 dilution

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: MYD88 knockout A549 cell lysate at 20 µg

  Lane 3: HEK-293 cell lysate at 20 µg

  Secondary

  Lanes 1 - 3: Goat anti-Mouse IgG H&L 680RD

  Lanes 1 - 3: Goat anti-Rabbit IgG H&L 800CW

  Performed under reducing conditions.

  Predicted band size: 33 kDa

  Observed band size: 35 kDa