Phospho-ULK1 (Ser555) Antibody [N1G15]

Catalog No.: F0305

Application: Reactivity: Human, Mouse

Lane 1: U-2 OS , Lane 2: U-2 OS (AMPK Activator, 50 μM, 1 hr) , Lane 3: MCF7 , Lane 4: MCF7 (oligomycin,0.5 μM,30 min)

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20ul	JPY 30400	国内在庫なし(納期7~10日)
100ul	JPY 67200	国内在庫あり
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代表番号: 045-509-1970\|電子メール：sales@selleck.co.jp よく尋ねられる質問

カスタマーフィードバック(1)

キーポイント

WB
SDS-PAGE の分離ゲルの推奨濃度：5%。
このタンパク質の基底発現は低いため、事前に誘導刺激を行うことを推奨します。

よくある問題とその対策

Fig. 1: WB (HepG2 Lysate) shows no bands (cells were not stimulated)

This antibody recognizes the Ser555-phosphorylated ULK1 protein. In some cell lines, the endogenous expression of Phospho-ULK1 (Ser555) is low without stimulation, leading to no visible bands. Therefore, it is recommended to stimulate the samples during preparation.

Fig. 2: WB (Human Pancreatic Cancer Cell Lysate) shows no bands (cells were not stimulated)

This antibody recognizes the Ser555-phosphorylated ULK1 protein, which is a key step in autophagy. In some cell lines, the endogenous expression of Phospho-ULK1 (Ser555) is low without stimulation. Therefore, it is recommended to stimulate the samples during preparation.

使用情報

Dilution
WB1:1000 IP1:100

Application
WB, IP

Source
Rabbit Monoclonal Antibody

Reactivity
Human, Mouse

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN₃

Storage (from the date of receipt)
–20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
140-150 kDa

ポジティブコントロール	U-2 OS; MCF-7 (treated with CCCP, 100 μM, 2 h); C2C12 (treated with hydrogen peroxide, 10 mM, 5 min)
ネガティブコントロール	U-2 OS (ULK1 KO); MCF-7; C2C12

サンプル処理データの例

サンプル	処理状況
U-2OS	AMPK Activator 991 (50 μM, 1 h)
MCF-7	Oligomycin (0.5 μM, 30 min)
MCF-7	Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (100 μM, 2 h)
C2C12	Hydrogen peroxide (10 mM, 5 min)
クリックして、さらに多くのサンプルデータを表示

^*異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

サンプル	処理状況

プロトコール

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes. 2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 120 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 36. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.

2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 120 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution ( recommending 5% BSA solution) for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

36. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

生物学的記述

Specificity
Phospho-ULK1 (Ser555) Antibody [N1G15] recognizes endogenous levels of total Phospho-ULK1 (Ser555) protein.

タンパク質の局在
細胞質

Uniprot ID
O75385

Clone
N1G15

Synonym(s)
Phospho-ULK1 (Ser555),ULK1 (phospho S555)

Background
Phospho-ULK1 (Ser555) is a crucial phosphorylation site within the ULK1 protein complex, pivotal for regulating autophagy, a cellular process crucial for maintaining metabolic balance. AMP-activated protein kinase (AMPK) phosphorylates Ser555 in response to energy stress like glucose deprivation, enhancing ULK1 activity and promoting autophagy initiation. This phosphorylation also facilitates mitophagy, the selective degradation of damaged mitochondria, in response to cellular energy signals. Alongside Ser555, ULK1 undergoes various nutrient-sensitive phosphorylation events by AMPK and the mechanistic target of rapamycin complex 1 (MTORC1), highlighting the complex interplay between nutrient availability and autophagy regulation.

Background

Phospho-ULK1 (Ser555) is a crucial phosphorylation site within the ULK1 protein complex, pivotal for regulating autophagy, a cellular process crucial for maintaining metabolic balance. AMP-activated protein kinase (AMPK) phosphorylates Ser555 in response to energy stress like glucose deprivation, enhancing ULK1 activity and promoting autophagy initiation. This phosphorylation also facilitates mitophagy, the selective degradation of damaged mitochondria, in response to cellular energy signals. Alongside Ser555, ULK1 undergoes various nutrient-sensitive phosphorylation events by AMPK and the mechanistic target of rapamycin complex 1 (MTORC1), highlighting the complex interplay between nutrient availability and autophagy regulation.

References
[1] Tian W, et al. FEBS Lett. 2015;589(15):1847-1854.

PVDFメンブレン孔径参考表

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%