Anti-ULK1 (phospho S757) antibody [EPR22265-9] - BSA and Azide free

• IP

Unknown

Immunoprecipitation - Anti-ULK1 (phospho S757) antibody [EPR22265-9] - BSA and Azide free (AB256537)

This data was developed using ab229909, the same antibody clone in a different buffer formulation.

ULK1 (phospho S757) was immunoprecipitated from 0.35 mg MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate with ab229909 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab229909 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution. Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.

Lane 1 : MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate, 10μg.

Lane 2 : ab229909 IP in MEF whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab229909 in MEF whole cell lysate.

Blocking/dilution buffer : 5% NFDM/TBST.

Exposure time : 3 mins.

All lanes:

Immunoprecipitation - Anti-ULK1 (phospho S757) antibody [EPR22265-9] (<a href='/en-us/products/primary-antibodies/ulk1-phospho-s757-antibody-epr22265-9-ab229909'>ab229909</a>) at 1/1000 dilution

Lane 1:

MEF (mouse embryonic fibroblast (immortalized)) whole cell lysate at 10 µg

Lane 2:

<a href='/en-us/products/primary-antibodies/ulk1-phospho-s757-antibody-epr22265-9-ab229909'>ab229909</a> IP in MEF whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of <a href='/en-us/products/primary-antibodies/ulk1-phospho-s757-antibody-epr22265-9-ab229909'>ab229909</a> in MEF whole cell lysate

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: 150 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-ULK1 (phospho S757) antibody [EPR22265-9] - BSA and Azide free (AB256537)

This data was developed using ab229909, the same antibody clone in a different buffer formulation.

Phosphorylation level of S757 is reduced during starvation (PMID : 28835610, 21258367). This blot was developed using a higher sensitivity ECL substrate.

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

Exposure time : 59 seconds.

All lanes:

Western blot - Anti-ULK1 (phospho S757) antibody [EPR22265-9] (<a href='/en-us/products/primary-antibodies/ulk1-phospho-s757-antibody-epr22265-9-ab229909'>ab229909</a>) at 1/1000 dilution

Lane 1:

Untreated MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate at 20 µg

Lane 2:

MEF cells were cultured in amino acid and serum-free, glucose-containing starvation buffer (EBSS) for 16 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>) at 1/100000 dilution

Observed band size: 150 kDa,50 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-ULK1 (phospho S757) antibody [EPR22265-9] - BSA and Azide free (AB256537)

This data was developed using ab229909, the same antibody clone in a different buffer formulation.

WB membrane in lane 2 was treated with alkaline phosphatase for 1h at 37°C. This blot was developed using a higher sensitivity ECL substrate.

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

Exposure time : 59 seconds.

All lanes:

Western blot - Anti-ULK1 (phospho S757) antibody [EPR22265-9] (<a href='/en-us/products/primary-antibodies/ulk1-phospho-s757-antibody-epr22265-9-ab229909'>ab229909</a>) at 1/1000 dilution

Lane 1:

MEF (mouse embryonic fibroblast (immortalized)), whole cell lysate at 20 µg

Lane 2:

MEF 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

Observed band size: 150 kDa,36 kDa

false

• Dot

Lab

Dot Blot - Anti-ULK1 (phospho S757) antibody [EPR22265-9] - BSA and Azide free (AB256537)

This data was developed using ab229909, the same antibody clone in a different buffer formulation.

Dot blot analysis using ab229909 at 1/1000 dilution, followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 diltution.

Lane 1 : ULK1 (phospho S757) peptide.

Lane 2 : ULK1 non-phospho peptide.

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

Exposure time : 3 minutes.