Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293 (human embryonic kidney epithelial cell) cells labelling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 (0.118 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing increased cytoplasmic and nuclear staining in HEK-293 cell line treated with Calyculin A (50 nM) for 3 h, then the signal decreased after phosphatase treatment at 37°C for 2h. is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2 ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HEK-293 (human embryonic kidney epithelial cell) treated with 50nM Calyculin A for 3 hours (Red) / Untreated control (Green) cells labelling Phospho - (Ser/Thr) Phe with ab300625 at 1/50 dilution (1ug) (Red) and Green (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 dilution (0.118 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining on human tonsil without alkaline phosphatase (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab300625 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) and used without primary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunohistochemical analysis of paraffin-embedded (A) HEK-293 (human e tissue labeling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 (0.118 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection) was used. Weak cytoplasmic staining on HEK-293 (human embryonic kidney epithelial cell) cell pellet (without alkaline phosphatase treatment) (image A). Nuclear and cytoplasmic staining on HEK-293 treated with 50nM Calyculin A for 3 hours cell pellet (without alkaline phosphatase treatment) (image B). No signal was detected HEK-293 treated with 50nM Calyculin A for 3 hours cell pellet (alkaline phosphatase treated) (image C). The section was incubated with ab300625 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 LeicaDS9800 (Bond™ Polymer Refine Detection).Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunohistochemical analysis of paraffin-embedded human hepatocellular tissue labeling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 dilution (0.118 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining on human hepatocellular carcinoma without alkaline phosphatase (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab300625 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) and added without primary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

• IP

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Immunoprecipitation - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Phospho - (Ser/Thr) Phe was immunoprecipitated from 0.35 mg HEK-293 (human embryonic kidney epithelial cell) treated with 50 nM Calyculin A for 3 hours, whole cell lysate with ab300625 at 1/30 dilution (2 µg in 0.35 mg lysates). Western blot was performed on the immunoprecipitate using ab300625 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Immunoprecipitation - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (ab300625) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) treated with 50nM Calyculin A for 3 hours, whole cell lysate at 10 µg

Lane 2:

ab300625 at 1/30 IP in HEK-293 treated with 50nM Calyculin A for 3 hours, whole cell lysate at 10 µg

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab300625 in HEK-293 treated with 50 nM Calyculin A for 3 hours, whole cell lysate at 10 µg

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

true

Exposure time: 3min

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 (0.118 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/Dab (ab209101). Nuclear and cytoplasmic staining was observed on rat testis without alkaline phosphatase (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab300625 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

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunohistochemical analysis of paraffin-embedded mouse lung tissue labeling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 dilution (0.118 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining was observed on mouse lung without alkaline phosphatase (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab300625 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) and used without primary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Phospho - (Ser/Thr) Phe with ab300625 at 1/4000 dilution (0.118 μg/mL), followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and cytoplasmic staining was observed on mouse testis without alkaline phosphatase (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab300625 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) and used without primary.

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20 mins was used.

• WB

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Western blot - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

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

Observed MW(kDa) : Multiple bands.

All lanes:

Western blot - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (ab300625) at 1/1000 dilution

Lane 1:

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

Lane 2:

HeLa treated with 100nM Calyculin A for 30 minutes, whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

HEK-293 treated with 50nM Calyculin A for 3 hours, 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>)

false

Exposure time: 103s

• WB

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Western blot - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

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

Observed MW(kDa) : Multiple bands.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

All lanes:

Western blot - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (ab300625) at 1/1000 dilution

Lane 1:

HEK-293 (human embryonic kidney epithelial cell) treated with 50nM Calyculin A for 3 hours, whole cell lysate at 20 µg

Lane 2:

HEK-293 treated with 50nM Calyculin A for 3 hours, whole cell lysate (phosphatase treated membrane) 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

true

Exposure time: 48s

• ELISA

Lab

ELISA - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Indirect ELISA antibody dose-response curve using ab300625 between 0-1000 ng/mL. Antigen concentration of 100 ng/mL. An alkaline phosphatase-conjugated AffiniPure goat anti-rabbit IgG (H+L) (1/2500) was used as the secondary antibody.

Phospho-(Ser/Thr) Phe peptides : a mixture of (Tyr/Phe/Trp)-Phospho - (Ser/Thr) Phe peptides.

• Dot

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Dot Blot - Anti-Phospho - (Ser/Thr) Phe antibody [EPR26858-4] (AB300625)

Dot blot analysis of Phospho - (Ser/Thr) Phe using ab300625 at 1 : 1000 (0.473 μg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 10,000 dilution. Lane 1 : Phospho - (Ser/Thr) Phe peptides Lane 2 : Non-phospho - (Ser/Thr) Phe peptides Lane 3 : Phospho - (Ser/Thr) (Lys/Arg/Asp/Glu) peptides Exposure time : 3 minutes Blocking and diluting buffer and concentration : 5% NFDM/TBST