Recombinant Anti-MyD88 antibody [EPR590(N)] (ab133739)

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: MyD88 knockout HAP1 cell lysate (20 µg)
Lane 3: Jurkat cell lysate (20 µg)
Lane 4: MOLT4 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab133739 observed at 37 kDa. Red - loading control, ab7291, observed at 52 kDa.

ab133739 was shown to specifically react with MyD88 when MyD88 knockout samples were used. Wild-type and MyD88 knockout samples were subjected to SDS-PAGE. ab133739 and ab7291 (loading control to alpha tubulin) were diluted 1/1000 and 1/10 000 respectively and incubated overnight at 4°C. 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 1/10000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing HAP1 wildtype (green line) and HAP1-MyD88 knockout cells (red line) stained with ab133739. The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab133739, 0.1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 22°C. A rabbit IgG1 isotype control antibody (ab172730) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-MyD88 knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity). Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. This antibody can also be used in HAP1 cells fixed with 4% formaldehyde (10 min) permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions. 

Blocking buffer: 5% NFDM/TBST

Dilution buffer: 5% NFDM/TBST

Immunofluorescence staining of Jurkat cells with purified ab133739 at a working dilution of 1/500, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab133739 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

Overlay histogram showing K562 cells fixed in 4% PFA and stained with purified ab133739 at a dilution of 1 in 350 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Overlay histogram showing MCF7 cells stained with unpurified ab133739 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab133739, 1/10000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

Immunohistochemistry analysis of Myd88 expression in formalin-fixed, paraffin-embedded Human kidney tissue, using unpurified ab133739 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.