Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded human testis tissue labelling PHF8 with ab280887 at 1/200 (1.066 μg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human testis. The section was incubated with ab280887 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

Supplier Data

Immunoprecipitation - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

PHF8 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) treated with 10μM MG-132 for 6 hours, whole cell lysate 10 ug with ab280887 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab280887 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) treated with 10μM MG-132 for 6 hours, whole cell lysate 10 ug

Lane 2 : ab280887 IP in HeLa treated with 10μM MG-132 for 6 hours, whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab280887 in HeLa treated with 10μM MG-132 for 6 hours, whole cell lysate

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

Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-PHF8 antibody [EPR23913-57] (<a href='/en-us/products/primary-antibodies/phf8-antibody-epr23913-57-ab280887'>ab280887</a>)

Predicted band size: 117 kDa

Observed band size: 135-140 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labelling PHF8 with ab280887 at 1/500 (1.066 μg/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse testis. The section was incubated with ab280887 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded rat colon tissue labelling PHF8 with ab280887 at 1/500 (1.066 μg/ml) dilution followed by ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat colon. The section was incubated with ab280887 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• WB

Lab

Western blot - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The observed MW is consistent with what has been described in the literature (PMID : 27183383).

Low expression : U-2 OS (PMID : 27183383 )

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 81 seconds

All lanes:

Western blot - Anti-PHF8 antibody [EPR23913-57] (<a href='/en-us/products/primary-antibodies/phf8-antibody-epr23913-57-ab280887'>ab280887</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

U-2 OS (human bone osteosarcoma 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 1/100000 dilution

Predicted band size: 117 kDa

Observed band size: 135-140 kDa

false

• WB

Lab

Western blot - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

False colour image of Western blot : Anti-PHF8 antibody [EPR23913-57] 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, ab280887 was shown to bind specifically to PHF8. A band was observed at 130 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in PHF8 knockout cell line ab280058 (knockout cell lysate ab280117). To generate this image, wild-type and PHF8 knockout HEK-293 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. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PHF8 antibody [EPR23913-57] (<a href='/en-us/products/primary-antibodies/phf8-antibody-epr23913-57-ab280887'>ab280887</a>) at 1/1000 dilution

Lane 1:

Wild-type HEK-293 cell lysate

Lane 2:

PHF8 knockout HEK-293 cell lysate

Lane 3:

Jurkat cell lysate

Lane 4:

HeLa cell lysate

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 130 kDa

false

• WB

Lab

Western blot - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The observed MW is consistent with what has been described in the literature (PMID : 25047223).

Exposure time : 158 seconds

All lanes:

Western blot - Anti-PHF8 antibody [EPR23913-57] (<a href='/en-us/products/primary-antibodies/phf8-antibody-epr23913-57-ab280887'>ab280887</a>) at 1/1000 dilution

All lanes:

Human cerebellum tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 117 kDa

Observed band size: 135-140 kDa

false

• WB

Lab

Western blot - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The observed MW is consistent with what has been described in the literature (PMID : 25047223).

Lysates (lane 3-5) were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : Lane 1-2 : 158 seconds; Lane 3-5 : 125 seconds

All lanes:

Western blot - Anti-PHF8 antibody [EPR23913-57] (<a href='/en-us/products/primary-antibodies/phf8-antibody-epr23913-57-ab280887'>ab280887</a>) at 1/1000 dilution

Lane 1:

Mouse testis tissue lysate at 20 µg

Lane 2:

Rat testis tissue lysate at 20 µg

Lane 3:

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

Lane 4:

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

Lane 5:

PC-12 (rat adrenal gland pheochromocytoma) 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: 117 kDa

Observed band size: 135-140 kDa

false

• WB

Lab

Western blot - Anti-PHF8 antibody [EPR23913-57] - BSA and Azide free (AB280896)

This data was developed using ab280887, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

This antibody has weak cross-reactivity with human PHF2.

These two recombinant proteins were made in house and expressed from E.coli expression systems.

All lanes:

Western blot - Anti-PHF8 antibody [EPR23913-57] (<a href='/en-us/products/primary-antibodies/phf8-antibody-epr23913-57-ab280887'>ab280887</a>) at 1/5000 dilution

Lane 1:

His-tagged human PHF8 recombinant protein at 10 µg

Lane 2:

His-tagged human PHF2 recombinant protein 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: 117 kDa

Observed band size: 135-140 kDa

false

Exposure time: 8s