Anti-MyD88 antibody [EPR590(N)]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MyD88 antibody [EPR590(N)] (AB133739)

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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MyD88 antibody [EPR590(N)] (AB133739)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MyD88 antibody [EPR590(N)] (AB133739)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MyD88 antibody [EPR590(N)] (AB133739)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-MyD88 antibody [EPR590(N)] (AB133739)

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 >5000 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MyD88 antibody [EPR590(N)] (AB133739)

Immunohistochemical staining of paraffin embedded human cerebral cortex with purified ab133739 at a working dilution of 1/500. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-MyD88 antibody [EPR590(N)] (ab133739)

Predicted band size: 33 kDa

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• WB

Lab

Western blot - Anti-MyD88 antibody [EPR590(N)] (AB133739)

Blocking buffer : 5% NFDM/TBST

Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-MyD88 antibody [EPR590(N)] (ab133739) at 1/5000 dilution

Lane 1:

HepG2 cell lysate at 29 µg

Lane 2:

K562 cell lysate at 29 µg

Lane 3:

Raji cell lysate at 29 µg

Lane 4:

Jurkat cell lysate at 29 µg

Secondary

All lanes:

HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 33 kDa

Observed band size: 33 kDa

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• WB

Lab

Western blot - Anti-MyD88 antibody [EPR590(N)] (AB133739)

Western blot : Anti-MYD88 antibody [EPR590(N)] (ab133739) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab133739 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 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 5% 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.

Lanes 1 - 3:

Western blot - Anti-MyD88 antibody [EPR590(N)] (ab133739) at 1/1000 dilution

Lanes 1 - 3:

Western blot - Anti-MyD88 antibody [EPR590(N)] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/myd88-antibody-epr590n-bsa-and-azide-free-ab199247'>ab199247</a>)

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

Observed band size: 35 kDa

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• WB

Supplier Data

Western blot - Anti-MyD88 antibody [EPR590(N)] (AB133739)

False colour image of Western blot : Anti-MyD88 antibody [EPR590(N)] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab133739 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 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.

All lanes:

Western blot - Anti-MyD88 antibody [EPR590(N)] (ab133739) 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 2:

Western blot - Human MYD88 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-myd88-knockout-a549-cell-line-ab286715'>ab286715</a>)

Lane 3:

HEK-293 cell lysate at 20 µg

Predicted band size: 33 kDa

Observed band size: 35 kDa

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• WB

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Western blot - Anti-MyD88 antibody [EPR590(N)] (AB133739)

All lanes:

Western blot - Anti-MyD88 antibody [EPR590(N)] (ab133739) at 1/1000 dilution

Lane 1:

Jurkat cell lysate at 10 µg

Lane 2:

Molt-4 cell lysate at 10 µg

Lane 3:

HepG2 cell lysate at 10 µg

Lane 4:

K562 cell lysate at 10 µg

Lane 5:

Raji cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit at 1/2000 dilution

Predicted band size: 33 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-MyD88 antibody [EPR590(N)] (AB133739)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.