Anti-YTHDF3 antibody [EPR21912-3]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling YTHDF3 with ab220161 at 1/60 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling YTHDF3 with ab220161 at 1/100 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on human colon (PMID : 29103884, 28250115) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IP

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Immunoprecipitation - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

YTHDF3 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate with ab220161 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220161 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used at 1/5000 dilution.

Lane 1 : HeLa whole cell lysate 10 μg (Input).
Lane 2 : ab220161 IP in HeLa whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab220161 in HeLa whole cell lysate.

Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 15 seconds.

Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

All lanes:

Immunoprecipitation - Anti-YTHDF3 antibody [EPR21912-3] (ab220161)

Predicted band size: 64 kDa

Observed band size: 73 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling YTHDF3 with ab220161 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on neurons of mouse cerebrum (PMID : 29103884, 28250115) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Immunohistochemical analysis of paraffin-embedded rat cerebrum tissue labeling YTHDF3 with ab220161 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Cytoplasmic staining on neurons of rat cerebrum (PMID : 29103884, 28250115) is observed. Counterstained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Immunohistochemistry analysis (Formalin/PFA-fixed paraffin-embedded sections) of paraformaldehyde-fixed mouse ovary tissue permeabilized with PBST (0.5% Triton X-100 in PBS). Stained with ab220161 at 1/200 dilution. Secondary antibody used was Alex Fluor 555 donkey anti-rabbit IgG at 1/500 dilution. Blocking was done with 5% BSA in PBST (0.1% tween in PBS) for 1 hour 30 minutes at 25°C. The sample was incubated with the primary antibody and 5% BSA in PBST (0.1% tween in PBS) for 12 hours at 4°C. Antigen retrieval method was heat mediated, Citrate antigen repair solution.

This image is courtesy of an Abreview submitted by Bin Shen

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling YTHDF3 with ab220161 at 1/100 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Cytoplasmic and nuclear staining in mouse cerebrum (PMID : 29103884, 28250115) is observed. Counterstained with DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

• IHC-Fr

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Immunohistochemistry (Frozen sections) - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen rat cerebrum tissue labeling YTHDF3 with ab220161 at 1/100 dilution (green), followed by ab150077 AlexaFluor^®488 Goat anti-Rabbit secondary at a 1/1,000 dilution. Cytoplasmic and nuclear staining in rat cerebrum (PMID : 29103884, 28250115) is observed. Counterstained with DAPI (blue).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is ab150077 AlexaFluor^®488 Goat anti-Rabbit used at a 1/1,000 dilution.

Heat mediated antigen retrieval using sodium citrate buffer (10 mM citrate pH 6.0 and 0.05% Tween-20).

• WB

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Western blot - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

Lysate should be made freshly and used in WB immediately to minimize protein degradation (lane1-4). Lane 5-7 are the lysates from same cell lines but have experienced freeze-thaw cycles.

Blocking/Dilution buffer : 5% NFDM/TBST.

Exposure times : Lanes 1-4 : 37 secs; Lanes 5-7 : 92 secs.

All lanes:

Western blot - Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/1000 dilution

Lane 1:

HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate (fresh lysate) at 20 µg

Lane 2:

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

Lane 3:

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

Lane 4:

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

Lane 5:

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

Lane 6:

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

Lane 7:

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: 64 kDa

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• WB

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Western blot - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

The wild-type and YTHDF3 knockout cell lysates were kindly provided by an anonymous collaborator.

ab220161 was shown to specifically react with YTHDF3 in wild-type mESC cells as signal was lost in YTHDF3 knockout cells. Wild-type and YTHDF3 knockout samples were subjected to SDS-PAGE. ab220161 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

The blot was developed on a BIO-RAD^® ChemiDoc™ MP instrument using the ECL technique.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/1000 dilution

Lane 1:

Wild-type mESC (mouse embryo stem cell) whole cell lysate at 20 µg

Lane 2:

YTHDF3 knockout mESC 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: 64 kDa

Observed band size: 73 kDa

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Exposure time: 59s

• WB

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Western blot - Anti-YTHDF3 antibody [EPR21912-3] (AB220161)

The YTHDF recombinant proteins were kindly provided by an anonymous collaborator.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF3 antibody [EPR21912-3] (ab220161) at 1/5000 dilution

Lane 1:

GST-tagged human YTHDF1 recombinant protein 20 ng

Lane 2:

GST-tagged human YTHDF2 recombinant protein 20 ng

Lane 3:

GST-tagged human YTHDF3 recombinant protein 20 ng

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: 64 kDa

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Exposure time: 10s