Anti-SMYD3 antibody [EPR19311]

• IP

Supplier Data

Immunoprecipitation - Anti-SMYD3 antibody [EPR19311] (AB199361)

SMYD3 was immunoprecipitated from 1mg of 293T (Human epithelial cell line from embryonic kidney) whole cell lysate with ab199361 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab199361 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : 293T whole cell lysate, 10μg (Input).

Lane 2 : ab199361 IP in 293T whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A ]-Isotype Control (ab172730) instead of ab199361 in 293T whole cell lysate.

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-SMYD3 antibody [EPR19311] (ab199361)

Predicted band size: 49 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-SMYD3 antibody [EPR19311] (AB199361)

SMYD3 was immunoprecipitated from 1mg of 293T (Human epithelial cell line from embryonic kidney) whole cell lysate with ab199361 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab199361 at 1/1000 dilution (Panel A) or ab183498 at 1/1000 dilution (Panel B).

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : 293T whole cell lysate, 10μg (Input).

Lane 2 : ab199361 IP in 293T whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab199361 in 293T whole cell lysate.

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

Exposure time : 3 minutes.

The product was validated with ab183498, which is specific to SMYD3.

All lanes:

Immunoprecipitation - Anti-SMYD3 antibody [EPR19311] (ab199361)

Predicted band size: 49 kDa

false

• WB

Lab

Western blot - Anti-SMYD3 antibody [EPR19311] (AB199361)

Lanes 1 - 4 : Merged signal (red and green). Green - ab199361 observed at 49 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab199361 was shown to react with SMYD3 in HAP1 wild-type cells in Western blot. Loss of signal was observed when SMYD3 knockout sample was used. HAP1 wild-type and SMYD3 knockout whole cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% Milk in TBS-T (0.1% Tween^®) before incubation with ab199361 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SMYD3 antibody [EPR19311] (ab199361) 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

Observed band size: 49 kDa

false

• WB

Supplier Data

Western blot - Anti-SMYD3 antibody [EPR19311] (AB199361)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SMYD3 antibody [EPR19311] (ab199361) at 1/2000 dilution

Lane 1:

Human fetal kidney lysate at 10 µg

Lane 2:

Human fetal brain lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 49 kDa

Observed band size: 45 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-SMYD3 antibody [EPR19311] (AB199361)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SMYD3 antibody [EPR19311] (ab199361) at 1/2000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

Lane 3:

T-47D (Human ductal breast epithelial tumor cell line) whole cell lysate at 20 µg

Lane 4:

HCT 116 (Human colorectal carcinoma cell line) whole cell lysate at 20 µg

Lane 5:

LLC (Mouse lung carcinoma cell line) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 49 kDa

Observed band size: 45 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-SMYD3 antibody [EPR19311] (AB199361)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-SMYD3 antibody [EPR19311] (ab199361) at 1/2000 dilution

Lane 1:

Mouse brain lysate at 10 µg

Lane 2:

Mouse kidney lysate at 10 µg

Lane 3:

Mouse spleen lysate at 10 µg

Lane 4:

Rat brain lysate at 10 µg

Lane 5:

Rat kidney lysate at 10 µg

Lane 6:

C6 (Rat glial tumor cell line) whole cell lysate at 10 µg

Lane 7:

RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate at 10 µg

Lane 8:

PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Lane 9:

NIH/3T3 (Mouse embryo fibroblast cell line) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 49 kDa

Observed band size: 45 kDa

false

Exposure time: 3min