Recombinant Anti-YTHDF2 antibody [EPR20318] (ab220163)

All lanes : Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/1000 dilution

Lane 1 : Wild-type mESC (mouse embryo stem cell) whole cell lysate
Lane 2 : YTHDF2 knockout mESC whole cell lysate
Lane 3 : HeLa (human cervix adenocarcinoma 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: 62 kDa


Exposure time: 48 seconds

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

The wild-type and YTHDF2 knockout cell lysates were kindly provided by an anonymous collaborator.

ab220163 was shown to specifically react with YTHDF2 in wild-type mESC cells as signal was lost in YTHDF2 knockout cells. Wild-type and YTHDF2 knockout samples were subjected to SDS-PAGE. ab220163 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.

ab220163 Immunoprecipitating YTHDF2 in human HepG2 whole cell lysate. 10 µg of cell lysate was incubated with primary antibody (1/30). For western blotting ab220163 (1/1000) was used to confirm successful immunoprecipitation. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at (1/5000). For lane 3 Rabbit monoclonal IgG (ab172730) was used instead of ab220163.

In panel A, the lysate was stored at -80^oC prior to the IP and incubation with the primary antibody was carried out overnight. In panel B, lysate was freshly made and used in the IP test immediately to minimize protein degradation. The incubation time was also shortened from overnight to 2h.

All lanes : Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/5000 dilution

Lane 1 : Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg
Lane 2 : HT-1080 treated with 10µM MG-132 (ab141003) for 4 hours, whole cell lysate at 10 µg

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

Predicted band size: 62 kDa


Exposure time: 103 seconds

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

All lanes : Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/1000 dilution

Lane 1 : Human brain tissue lysate
Lane 2 : Mouse testis tissue lysate
Lane 3 : Rat testis tissue lysate
Lane 4 : HEK-293 (human embryonic kidney epithelial cell) whole cell lysate
Lane 5 : K562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lane 1 : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution
Lanes 2-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 62 kDa

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

Exposure time: Lane 1: 3 minutes; Lane 2: 92 seconds; Lane 3: 37 seconds; Lane 4: 8 seconds; Lane 5: 15 seconds.

 

All lanes : Anti-YTHDF2 antibody [EPR20318] (ab220163) at 1/1000 dilution

Lane 1 : GST-tagged human YTHDF1 recombinant protein, 20ng
Lane 2 : GST-tagged human YTHDF2 recombinant protein, 20ng
Lane 3 : GST-tagged human YTHDF3 recombinant protein, 20ng

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

Predicted band size: 62 kDa


Exposure time: 3 seconds

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

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