Phospho-ULK1 (Ser638) Antibody (Rabbit mAb) [F14B19]

Application: Reactivity: Human, Mouse, Monkey

Lane 1: Hela

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Mouse, Monkey

Source
Rabbit Monoclonal Antibody

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
113 kDa

Positive Control	A172 cells; MCF7 cells; A20 cells; BaF3 cells
Negative Control

Experimental Methods

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 wash the cells with ice-cold PBS twice. Lyse the cells by adding 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. Lyse the cells by adding 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 primary antibody dilution buffer 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 1. 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.

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 wash the cells with ice-cold PBS twice. Lyse the cells by adding 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. Lyse the cells by adding 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 primary antibody dilution buffer 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

1. 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

Biological Description

Specificity
Phospho-ULK1 (Ser638) Antibody (Rabbit mAb) [F14B19] detects endogenous levels of ULK1 protein only when phosphorylated at Ser638.

Subcellular Location
Cytoplasm

Uniprot ID
O75385

Clone
F14B19

Synonym(s)
ATG1; ATG1 autophagy related 1 homolog; ATG1A; FLJ38455; FLJ46475; hATG1; KIAA0722; ULK1; unc-51-like kinase 1 (C. elegans); UNC51; Unc51.1

Background
Phosphorylated ULK1 at Ser638 represents a nutrient- and stress-regulated state of the UNC‑51-like kinase 1, an Atg1-family serine/threonine kinase that forms the core autophagy‑initiating ULK1 complex and links upstream mTORC1 and AMPK signaling to downstream autophagosome biogenesis, isolation membrane expansion and organelle clearance. ULK1 contains an N‑terminal kinase domain, a central serine/proline‑rich linker and a conserved C‑terminal domain that scaffolds ATG13, FIP200/RB1CC1 and ATG101, and multiple phosphorylation sites within the linker region integrate signals from mTORC1 and AMPK; Ser638 lies in this regulatory linker and is phosphorylated by mTORC1 under nutrient‑replete conditions in coordination with Ser757, as well as being targeted by AMPK under certain stress contexts, establishing Ser638 as a bidirectionally regulated site that participates in switching ULK1 between inactive and active conformations. Under anabolic, growth‑factor‑rich conditions, active mTORC1 associates with the ULK1 complex and phosphorylates ULK1 at Ser638 and Ser757 and ATG13 at mTOR‑sensitive sites, events that reduce ULK1 kinase activity, disrupt ULK1–AMPK interaction and retain the complex at non‑initiating locations, thereby suppressing autophagy initiation; upon acute nutrient starvation or energy stress, mTORC1 is inhibited and dissociates, Ser638 and related inhibitory sites undergo dephosphorylation, and AMPK is activated and directly phosphorylates ULK1 on pro‑autophagic sites (Ser317, Ser555, Ser777), promoting ULK1 complex relocalization to pre‑autophagosomal membranes and induction of autophagy. Recent analyses of the Atg1/ULK1 regulatory mechanism indicate that ULK1 phosphorylates ATG9 to control isolation membrane expansion and that Ser638 phosphorylation participates in tuning this activity: mTORC1‑mediated phosphorylation of Ser638 and neighboring residues reduces ULK1‑dependent ATG9 phosphorylation and limits phagophore growth, whereas AMPK‑linked regulation of Ser638 associates with enhanced ULK1 activity toward ATG9 and increased autophagosome formation in response to stress, placing phospho‑Ser638 at a key junction between nutrient sensing and membrane expansion control. ULK1 also phosphorylates substrates beyond the initiation complex, including Beclin‑1 in the VPS34 class III PI3K complex and the mitophagy receptor BNIP3, and phospho‑state profiling shows that Ser638 is part of a broader phospho‑code that modulates ULK1 substrate preference and its transition from autophagosome nucleation to maturation and organelle‑selective pathways such as mitophagy. In cancer, ULK1‑mediated autophagy contributes to drug resistance by supporting metabolic flexibility, stress adaptation and survival under hypoxia and nutrient limitation, and reviews of ULK1 in therapy resistance emphasize that mTORC1 and AMPK activity, acting through ULK1 linker sites including Ser638, shape whether autophagy is suppressed or engaged during treatment; pharmacologic targeting of ULK1 or its phospho‑regulation, including modulation of Ser638 phosphorylation, is under investigation as a strategy to sensitize tumors to chemotherapy and radiotherapy.

Background

Phosphorylated ULK1 at Ser638 represents a nutrient- and stress-regulated state of the UNC‑51-like kinase 1, an Atg1-family serine/threonine kinase that forms the core autophagy‑initiating ULK1 complex and links upstream mTORC1 and AMPK signaling to downstream autophagosome biogenesis, isolation membrane expansion and organelle clearance. ULK1 contains an N‑terminal kinase domain, a central serine/proline‑rich linker and a conserved C‑terminal domain that scaffolds ATG13, FIP200/RB1CC1 and ATG101, and multiple phosphorylation sites within the linker region integrate signals from mTORC1 and AMPK; Ser638 lies in this regulatory linker and is phosphorylated by mTORC1 under nutrient‑replete conditions in coordination with Ser757, as well as being targeted by AMPK under certain stress contexts, establishing Ser638 as a bidirectionally regulated site that participates in switching ULK1 between inactive and active conformations. Under anabolic, growth‑factor‑rich conditions, active mTORC1 associates with the ULK1 complex and phosphorylates ULK1 at Ser638 and Ser757 and ATG13 at mTOR‑sensitive sites, events that reduce ULK1 kinase activity, disrupt ULK1–AMPK interaction and retain the complex at non‑initiating locations, thereby suppressing autophagy initiation; upon acute nutrient starvation or energy stress, mTORC1 is inhibited and dissociates, Ser638 and related inhibitory sites undergo dephosphorylation, and AMPK is activated and directly phosphorylates ULK1 on pro‑autophagic sites (Ser317, Ser555, Ser777), promoting ULK1 complex relocalization to pre‑autophagosomal membranes and induction of autophagy. Recent analyses of the Atg1/ULK1 regulatory mechanism indicate that ULK1 phosphorylates ATG9 to control isolation membrane expansion and that Ser638 phosphorylation participates in tuning this activity: mTORC1‑mediated phosphorylation of Ser638 and neighboring residues reduces ULK1‑dependent ATG9 phosphorylation and limits phagophore growth, whereas AMPK‑linked regulation of Ser638 associates with enhanced ULK1 activity toward ATG9 and increased autophagosome formation in response to stress, placing phospho‑Ser638 at a key junction between nutrient sensing and membrane expansion control. ULK1 also phosphorylates substrates beyond the initiation complex, including Beclin‑1 in the VPS34 class III PI3K complex and the mitophagy receptor BNIP3, and phospho‑state profiling shows that Ser638 is part of a broader phospho‑code that modulates ULK1 substrate preference and its transition from autophagosome nucleation to maturation and organelle‑selective pathways such as mitophagy. In cancer, ULK1‑mediated autophagy contributes to drug resistance by supporting metabolic flexibility, stress adaptation and survival under hypoxia and nutrient limitation, and reviews of ULK1 in therapy resistance emphasize that mTORC1 and AMPK activity, acting through ULK1 linker sites including Ser638, shape whether autophagy is suppressed or engaged during treatment; pharmacologic targeting of ULK1 or its phospho‑regulation, including modulation of Ser638 phosphorylation, is under investigation as a strategy to sensitize tumors to chemotherapy and radiotherapy.

References
https://pubmed.ncbi.nlm.nih.gov/26921696/ https://pubmed.ncbi.nlm.nih.gov/21258367/

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%