Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free

• IP

Unknown

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (AB253038)

YTHDF1 was immunoprecipitated from 0.35 mg HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate with ab220162 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220162 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.
Lane 1 : HepG2 whole cell lysate 10 μg (Input).
Lane 2 : ab220162 IP in HepG2 whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab220162 in HepG2 whole cell lysate.
Blocking/Dilution buffer : 5% NFDM/TBST.
Exposure time : 3 seconds.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab220162).

All lanes:

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] (<a href='/en-us/products/primary-antibodies/ythdf1-antibody-epr22349-41-ab220162'>ab220162</a>)

Predicted band size: 61 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (AB253038)

This data was developed using ab220162, the same antibody clone in a different buffer formulation. YTHDF1 was immunoprecipitated from HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate with ab220162 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab220162 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution. Lane 1 : HeLa whole cell lysate 10 μg (Input). Lane 2 : ab220162 IP in HeLa whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab220162 in HeLa whole cell lysate. Blocking/Dilution buffer : 5% NFDM/TBST. Lysate was freshly made and used for Western blotting immediately to minimize protein degradation.

All lanes:

Immunoprecipitation - Anti-YTHDF1 antibody [EPR22349-41] (<a href='/en-us/products/primary-antibodies/ythdf1-antibody-epr22349-41-ab220162'>ab220162</a>) at 1/1000 dilution

All lanes:

HeLa whole cell lysate at 10 µg

Predicted band size: 61 kDa

Observed band size: 60 kDa

false

Exposure time: 8s

• WB

Unknown

Western blot - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (AB253038)

This data was developed using ab220162, the same antibody clone in a different buffer formulation.

The wild-type and YTHDF1 knockout cell lysates were kindly provided by an anonymous collaborator.

ab220162 was shown to specifically react with YTHDF1 in wild-type mESC cells as signal was lost in YTHDF1 knockout cells. Wild-type and YTHDF1 knockout samples were subjected to SDS-PAGE. ab220162 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.

Exposure times : Lanes 1 and 2 : 59 seconds; Lanes 3 and 4 : 26 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (ab253038) at 1/1000 dilution

Lane 1:

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

Lane 2:

YTHDF1 knockout mESC whole cell lysate at 20 µg

Lane 3:

HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma 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: 61 kDa

Observed band size: 61 kDa

false

• WB

Supplier Data

Western blot - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (AB253038)

This data was developed using ab220162, the same antibody clone in a different buffer formulation. Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. Blocking and diluting buffer and concntration : 5% NFDM/TBST Imaging carried out using iBright CL 1000 Imaging System

All lanes:

Western blot - Anti-YTHDF1 antibody [EPR22349-41] (<a href='/en-us/products/primary-antibodies/ythdf1-antibody-epr22349-41-ab220162'>ab220162</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

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

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma 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: 61 kDa

Observed band size: 60 kDa

false

Exposure time: 180s

• WB

Unknown

Western blot - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (AB253038)

This data was developed using ab220162, the same antibody clone in a different buffer formulation.

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

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-YTHDF1 antibody [EPR22349-41] - BSA and Azide free (ab253038) at 1/1000 dilution

Lane 1:

GST-tagged human YTHDF1 recombinant protein at 20 µg

Lane 2:

GST-tagged human YTHDF2 recombinant protein at 20 µg

Lane 3:

GST-tagged human YTHDF3 recombinant protein 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: 61 kDa

Observed band size: 87 kDa

false

Exposure time: 3s