Anti-TAF15 antibody [EPR9197(B)]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

Overlay histogram showing HeLa cells stained with ab134916 (red line). The cells were fixed with 80% methanol (5 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 (ab134916, 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

Immunofluorescent analysis of TAF15 in HeLa cells, using ab134916 at a 1/250 dilution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

Immunohistochemical analysis using ab134916 showing positive staining in Normal human uterus tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

ab134916 staining TAF15 in HeLa (human cervix adenocarcinoma) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000. DAPI was used as a nuclear counterstain.

Negative control 1 : PBS only.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

Immunohistochemical analysis of TAF15 in paraffin embedded Human urinary bladder carcinoma tissue, using ab134916 at a 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• WB

Lab

Western blot - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

This blot was produced using 4-20% SDS-PAGE containing 15 μg of Caco-2 whole cell lysate per lane at 150V for 1hr before being transferred onto a 0.45 μm PVDF membrane at 75V for 1hr. The membrane was then blocked for 1hr using 5% NFDM/TBST, then incubated with ab134916 (1/10,000) at room temperature for 1hr. After being washed three times in TBST, the membrane was incubated with Peroxidase conjugated goat anti-rabbit IgG (H+L) (ab97051) at 1/20,000 dilution for 1hr at room temperature. The membrane was washed three times again. Then the signal was developed using the ECL technique. ab134916 was stored at a range of temperatures (+4°C, +22°C, +37°C) for 1 week before being tested in WB. The image shows the band intensity remains relatively constant across all storage temperatures, demonstrating that antibody activity is not affected.

All lanes:

Western blot - Anti-TAF15 antibody [EPR9197(B)] (ab134916) at 1/10000 dilution

All lanes:

Caco-2 whole cell lysate at 15 µg with NDFM/TBST

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

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Exposure time: 80s

• WB

Lab

Western blot - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

Western blot : Anti-TAF15 antibody [EPR9197(B)] ab134916 staining at 1/10000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 74 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in TAF15 knockout U-87 MG 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 and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-TAF15 antibody [EPR9197(B)] (ab134916) at 1/10000 dilution

Lane 1:

Wild-type U-87 MG whole cell lysate at 20 µg

Lane 2:

TAF15 knockout U-87 MG whole cell lysate at 20 µg

Lane 3:

Raji whole cell lysate at 20 µg

Lane 4:

HT1080 whole cell lysate at 20 µg

Lane 5:

PC-3 whole cell lysate at 20 µg

Secondary

All lanes:

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

Predicted band size: 62 kDa

Observed band size: 74 kDa

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• WB

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Western blot - Anti-TAF15 antibody [EPR9197(B)] (AB134916)

All lanes:

Western blot - Anti-TAF15 antibody [EPR9197(B)] (ab134916) at 1/10000 dilution

Lane 1:

HeLa cell lysate at 10 µg

Lane 2:

293T cell lysate at 10 µg

Lane 3:

Caco 2 cell lysate at 10 µg

Lane 4:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 62 kDa

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