Anti-POT1a antibody [EPR30481-543]

• IP

Lab

Immunoprecipitation - Anti-POT1a antibody [EPR30481-543] (AB325274)

Pot1a was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate with ab325274 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325274 at 1/325274 1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 2 : ab325274 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab325274 in NIH/3T3 whole cell lysate

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

Exposure time : 180 seconds.

To minimize protein degradation, cells were lysed immediately after harvest and then applied for Immunoprecipitation as soon as possible.

All lanes:

Immunoprecipitation - Anti-POT1a antibody [EPR30481-543] (ab325274)

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 10 µg

Lane 2:

ab325274 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 1/30 dilution

Lane 3:

Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>)

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

Observed band size: 71 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-POT1a antibody [EPR30481-543] (AB325274)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-POT1a antibody [EPR30481-543] (ab325274) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

NIH/3T3 transfected with siRNA specifically targeting Pot1a 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 1/20000 dilution

Observed band size: 71 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-POT1a antibody [EPR30481-543] (AB325274)

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

This antibody does not cross-react with mouse Pot1b.

In Western blot, Anti-6XHis tag® antibody [EPR20547] - ChIP Grade (ab213204) (1 : 5000)(71KDa); Anti-Histone H3 antibody [EPR16987] - Nuclear Marker and ChIP Grade (ab176842) 1 : 100000 (15Kda).

All lanes:

Western blot - Anti-POT1a antibody [EPR30481-543] (ab325274) at 1/1000 dilution

Lane 1:

293T cells transfected with an empty vector containing a His-tag, whole cell lysate at 20 µg

Lane 2:

293T cells transfected with a mouse Pot1a expression vector containing a His-tag, whole cell lysate at 20 µg

Lane 3:

293T cells transfected with a mouse Pot1b expression vector containing a His-tag, 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

Observed band size: 71 kDa,15 kDa

false

Exposure time: 6s

• WB

Lab

Western blot - Anti-POT1a antibody [EPR30481-543] (AB325274)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-POT1a antibody [EPR30481-543] (ab325274) at 1/1000 dilution

Lane 1:

Mouse liver tissue lysate at 20 µg

Lane 2:

Mouse lung tissue lysate at 20 µg

Lane 3:

Mouse testis tissue lysate at 20 µg

Lane 4:

Mouse kidney tissue 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 1/20000 dilution

Observed band size: 71 kDa,36 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-POT1a antibody [EPR30481-543] (AB325274)

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-POT1a antibody [EPR30481-543] (ab325274) at 1/1000 dilution

Lane 1:

MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 2:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 3:

F9 (mouse embryonal carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

CT26.WT (mouse colon fibroblast) 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 1/20000 dilution

Observed band size: 71 kDa,36 kDa

false

Exposure time: 37s