Anti-TRIM8 antibody [EPR27965-6]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-TRIM8 antibody [EPR27965-6] (AB316149)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HCT 116 (human colorectal carcinoma epithelial cell) cells labelling TRIM8 with ab316149 at 1/50 dilution (1 ug)/(Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-TRIM8 antibody [EPR27965-6] (AB316149)

TRIM8 was immunoprecipitated from 0.35 mg HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate with ab316149 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab316149 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 2 : ab316149 IP in HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab316149 in HCT 116 whole cell lysate

All lanes:

Immunoprecipitation - Anti-TRIM8 antibody [EPR27965-6] (ab316149) at 1/30 dilution

All lanes:

HCT 116 (human colorectal carcinoma epithelial cell) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 102s

• WB

Supplier Data

Western blot - Anti-TRIM8 antibody [EPR27965-6] (AB316149)

Lysates were freshly made and used for Western blotting immediately to minimize protein degradation.

The identity of the higher MW band at approximately 90 kDa (in lane 4) is unknown.

All lanes:

Western blot - Anti-TRIM8 antibody [EPR27965-6] (ab316149) at 1/1000 dilution

Lane 1:

HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lanes 6 - 7:

T-47D (human ductal breast epithelial tumor epithelial cell) 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 1/20000 dilution

Observed band size: 61 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-TRIM8 antibody [EPR27965-6] (AB316149)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-TRIM8 antibody [EPR27965-6] (ab316149) at 1/1000 dilution

Lane 1:

HCT 116 (human colorectal carcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

HCT 116 transfected with siRNA specifically targeting TRIM8 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 1/20000 dilution

Observed band size: 61 kDa,36 kDa

false

Exposure time: 92s

• WB

Lab

Western blot - Anti-TRIM8 antibody [EPR27965-6] (AB316149)

Western blot : Anti-TRIM8 antibody [EPR27965-6] ab316149 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 65 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in TRIM8 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-TRIM8 antibody [EPR27965-6] (ab316149) at 1/1000 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

TRIM8 knockout MCF7 at 20 µg

Lane 3:

OVCAR-3 at 20 µg

Lane 4:

HCT 116 at 20 µg

Lane 5:

HepG2 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 65 kDa

Observed band size: 65 kDa

false

• WB

Supplier Data

Western blot - Anti-TRIM8 antibody [EPR27965-6] (AB316149)

All lanes:

Western blot - Anti-TRIM8 antibody [EPR27965-6] (ab316149) at 1/1000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Lane 3:

Rat spleen 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 1/20000 dilution

Observed band size: 61 kDa

false

Exposure time: 180s