Anti-SMYD3 antibody [EPR11106(2)] - N-terminal

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

Intracellular flow cytometric analysis of 293 cells fixed in 2% paraformaldehyde labeling SMYD3 with ab183498 at 1/110 dilution andGoat anti rabbit IgG (FITC) at 1/150 dilution. Rabbit Monoclonal IgG was used as an isotype control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

Immunohistochemical analysis of paraffin-embedded Human testis labeling SMYD3 with ab183498 at 1/100 dilution and HRP Polymer for Rabbit IgG. Counterstained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

Flow cytometric analysis of HeLa cells fixed in 20 °C acetone labeling SMYD3 at 1/500 dilution and Goat anti rabbit IgG(Alexa Fluor® 555) at 1/200 (red). Counterstained with DAPI (blue)

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

Immunohistochemical analysis of paraffin-embedded Human skeletal muscle labeling SMYD3 at 1/100 dilution and HRP Polymer for Rabbit IgG. Counterstained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• IP

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Immunoprecipitation - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

Immunoprecipitation of HeLa cells labeling SMYD3 with ab183498 at 1/50 dilution and Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1500.

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Immunoprecipitation - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (ab183498)

Predicted band size: 49 kDa

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• WB

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Western blot - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

All lanes:

Western blot - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (ab183498) at 1/10000 dilution

Lane 1:

HeLa lysate at 20 µg

Lane 2:

293 lysate at 20 µg

Lane 3:

T47-D lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 49 kDa

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• WB

Lab

Western blot - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

Lanes 1 - 4 : Merged signal (red and green). Green - ab183498 observed at 49 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab183498 was shown to recognize SMYD3 in wild-type HAP1 cells as signal was lost at the expected MW in SMYD3 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SMYD3 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. ab183498 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (ab183498) at 1/1000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

SMYD3 knockout HAP1 whole cell lysate at 20 µg

Lane 3:

HeLa whole cell lysate at 20 µg

Lane 4:

MCF7 whole cell lysate at 20 µg

Predicted band size: 49 kDa

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• WB

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Western blot - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (AB183498)

All lanes:

Western blot - Anti-SMYD3 antibody [EPR11106(2)] - N-terminal (ab183498) at 1/2000 dilution

All lanes:

MCF-7 lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 49 kDa

false