All lanes : Anti-PHF8 antibody [EPR23913-57] (ab280887) at 1/1000 dilution

Lane 1 : HEK-293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 2 : U-2 OS (human bone osteosarcoma epithelial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 117 kDa
Observed band size: 135/140 kDa why is the actual band size different from the predicted?

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

 

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.

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

 

Anti-PHF8 antibody [EPR23913-57] (ab280887) at 1/1000 dilution + Human cerebellum tissue lysate at 20 µg

Secondary
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 why is the actual band size different from the predicted?

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 : Anti-PHF8 antibody [EPR23913-57] (ab280887) at 1/1000 dilution

Lane 1 : Mouse testis tissue lysate
Lane 2 : Rat testis tissue lysate
Lane 3 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 5 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 117 kDa
Observed band size: 135/140 kDa why is the actual band size different from the predicted?

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 : Anti-PHF8 antibody [EPR23913-57] (ab280887) at 1/5000 dilution

Lane 1 : His-tagged human PHF8 recombinant protein
Lane 2 : His-tagged human PHF2 recombinant protein

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 117 kDa
Observed band size: 135/140 kDa why is the actual band size different from the predicted?

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.

Exposure time: 8 seconds

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.

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.