Phospho-MLKL (Ser358) Antibody [K9E15]

Application: Reactivity: Human

Lane 1: HT-29, Lane 2: HT-29 (TNFα, 20ng/ml; Calyculin A, 100nM, 0.5 h)

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human

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
54 kDa

Positive Control	HT-29 cells (Z-VAD, 20 μM, added 30 min prior to other cpds; hTNF-α, 20 ng/ml, 7 h; SM-164, 100 nM, 7 h)
Negative Control	HT-29 cells

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: 10%. 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: 10%. 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-MLKL (Ser358) Antibody (Rabbit mAb) [K9E15] detects endogenous levels of total MLKL protein only when it is phosphorylated at Ser358.

Subcellular Location
Cell membrane, Cytoplasm, Membrane

Uniprot ID
Q8NB16

Clone
K9E15

Synonym(s)
FLJ34389, hMLKL, mixed lineage kinase domain like pseudokinase, mixed lineage kinase domain-like, Mixed lineage kinase domain-like protein, MLKL

Background
Phospho‑MLKL (Ser358) represents the activated executioner form of the mixed lineage kinase domain‑like pseudokinase within the necroptosis pathway, where MLKL acts downstream of RIPK3 to convert receptor‑proximal necrosome signaling into terminal membrane disruption and lytic cell death. The full‑length protein contains an N‑terminal four‑helix bundle executioner domain, a central brace region, and a C‑terminal pseudokinase domain whose activation loop harbors Thr357 and Ser358; this pseudokinase segment does not catalyze phosphate transfer but functions as a regulatory switch that senses phosphorylation by RIPK3 and allosterically releases the N‑terminal helical bundle to engage membranes. Necroptotic signaling initiated by death receptors, pattern‑recognition receptors, or other upstream inputs converges on RIPK3 activation and assembly of RIPK1–RIPK3–MLKL complexes, where RIPK3 phosphorylates the MLKL activation loop at Thr357 and Ser358, generating the phospho‑MLKL (Ser358) species that correlates with commitment to necroptotic execution. Phosphorylation at these residues drives a conformational transition in the pseudokinase domain that promotes dissociation of MLKL from RIPK3, exposure of the N‑terminal four‑helix bundle, and formation of higher‑order MLKL oligomers that translocate from the cytosol to the plasma membrane. Oligomerized, phospho‑MLKL inserts its N‑terminal helices into the inner leaflet of the plasma membrane and associates with specific phospholipids, where it perturbs membrane organization and integrity and forms disruptive assemblies that cause ion imbalance, swelling, and eventual membrane rupture characteristic of necroptotic cell death. Structural and biochemical analyses indicate that activation loop phosphorylation is sufficient to license MLKL killing activity, and phosphomimetic activation‑loop mutants bypass upstream signals to induce stimulus‑independent necrosis, underscoring the central regulatory position of the Thr357/Ser358 region and supporting the use of Ser358 phosphorylation as a precise molecular marker of MLKL activation state. Post‑translational modifications on MLKL beyond the activation loop, including additional phosphorylation events within the pseudokinase domain, further tune oligomerization efficiency, membrane engagement, and subcellular localization, placing Ser358 phosphorylation within a broader modification code that controls the amplitude and context of MLKL‑dependent necroptosis in infection and inflammatory settings. Phospho‑MLKL (Ser358) localizes predominantly to the plasma membrane and, in some contexts, to intracellular membranes at late stages of necroptosis, distinguishing it from nonphosphorylated MLKL and providing a spatial and biochemical readout of execution‑phase necroptotic signaling that is widely used to map necroptotic activity in tissues and disease models. In pathophysiological conditions, including ischemic injury, inflammatory diseases, and certain cancers, accumulation of phospho‑MLKL (Ser358) accompanies RIPK3 activation and necroptotic cell loss, linking this modification to tissue damage, inflammatory cytokine release, and modulation of the tumor microenvironment, while genetic or pharmacologic suppression of the RIPK3–MLKL axis reduces phospho‑MLKL formation and necroptotic output.

Background

Phospho‑MLKL (Ser358) represents the activated executioner form of the mixed lineage kinase domain‑like pseudokinase within the necroptosis pathway, where MLKL acts downstream of RIPK3 to convert receptor‑proximal necrosome signaling into terminal membrane disruption and lytic cell death. The full‑length protein contains an N‑terminal four‑helix bundle executioner domain, a central brace region, and a C‑terminal pseudokinase domain whose activation loop harbors Thr357 and Ser358; this pseudokinase segment does not catalyze phosphate transfer but functions as a regulatory switch that senses phosphorylation by RIPK3 and allosterically releases the N‑terminal helical bundle to engage membranes. Necroptotic signaling initiated by death receptors, pattern‑recognition receptors, or other upstream inputs converges on RIPK3 activation and assembly of RIPK1–RIPK3–MLKL complexes, where RIPK3 phosphorylates the MLKL activation loop at Thr357 and Ser358, generating the phospho‑MLKL (Ser358) species that correlates with commitment to necroptotic execution. Phosphorylation at these residues drives a conformational transition in the pseudokinase domain that promotes dissociation of MLKL from RIPK3, exposure of the N‑terminal four‑helix bundle, and formation of higher‑order MLKL oligomers that translocate from the cytosol to the plasma membrane. Oligomerized, phospho‑MLKL inserts its N‑terminal helices into the inner leaflet of the plasma membrane and associates with specific phospholipids, where it perturbs membrane organization and integrity and forms disruptive assemblies that cause ion imbalance, swelling, and eventual membrane rupture characteristic of necroptotic cell death. Structural and biochemical analyses indicate that activation loop phosphorylation is sufficient to license MLKL killing activity, and phosphomimetic activation‑loop mutants bypass upstream signals to induce stimulus‑independent necrosis, underscoring the central regulatory position of the Thr357/Ser358 region and supporting the use of Ser358 phosphorylation as a precise molecular marker of MLKL activation state. Post‑translational modifications on MLKL beyond the activation loop, including additional phosphorylation events within the pseudokinase domain, further tune oligomerization efficiency, membrane engagement, and subcellular localization, placing Ser358 phosphorylation within a broader modification code that controls the amplitude and context of MLKL‑dependent necroptosis in infection and inflammatory settings. Phospho‑MLKL (Ser358) localizes predominantly to the plasma membrane and, in some contexts, to intracellular membranes at late stages of necroptosis, distinguishing it from nonphosphorylated MLKL and providing a spatial and biochemical readout of execution‑phase necroptotic signaling that is widely used to map necroptotic activity in tissues and disease models. In pathophysiological conditions, including ischemic injury, inflammatory diseases, and certain cancers, accumulation of phospho‑MLKL (Ser358) accompanies RIPK3 activation and necroptotic cell loss, linking this modification to tissue damage, inflammatory cytokine release, and modulation of the tumor microenvironment, while genetic or pharmacologic suppression of the RIPK3–MLKL axis reduces phospho‑MLKL formation and necroptotic output.

References
https://pubmed.ncbi.nlm.nih.gov/33064829/ https://pubmed.ncbi.nlm.nih.gov/24703947/

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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Handling Instructions

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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%

Phospho-MLKL (Ser358) Antibody (Rabbit mAb) [K9E15]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support