Phospho-Vimentin (Ser56) Antibody [M10H3]

Application: Reactivity: Human, Mouse, Rat, Monkey

Lane 1: Hela (Hydroxyurea, 4 mM, G1/S arrested, 20 h), Lane 2: Hela (Paclitaxel, 100 nM, G2/M arrested, 20 h)

Usage Information

Dilution
WB1:1000 IF1:200

Application
WB, IF

Reactivity
Human, Mouse, Rat, 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 Observed MW
54 kDa 57 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Positive Control	HeLa cells (hydroxyurea, 4 mM, G1/S arrested); C2C12 cells (hydroxyurea, 4 mM, G1/S arrested)
Negative Control	HeLa cells (Paclitaxel, 100 nM, 20 h, G2/M arrested); C2C12 cells (Paclitaxel, 100 nM, 20 h, G2/M arrested); MCF7 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.

IF
Experimental Protocol: Sample Preparation 1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use. 2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine. 3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time. 4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval. Fixation 1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes. 2. Wash the sample with PBS for 3 times, 3 minutes each time. Permeabilization 1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes. (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.) Wash the sample with PBS for 3 times, 3 minutes each time. Blocking Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.) Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background. Immunofluorescence Staining (Day 1) 1. Remove the blocking solution and add the diluted primary antibody. 2. Incubate the sample in a humidified chamber at 4°C overnight. Immunofluorescence Staining (Day 2) 1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time. 2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours. 3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time. 4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes. 5. Wash with PBST for 3 times, 5 minutes each time. Mounting 1. Mount the sample with an anti-fade mounting medium. 2. Allow the slide to dry at room temperature overnight in the dark. 3. Store the slide in a slide storage box at 4°C, protected from light.

IF

Experimental Protocol:

Sample Preparation

1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.

2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.

3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.

4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.

Fixation

1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.

2. Wash the sample with PBS for 3 times, 3 minutes each time.

Permeabilization

1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.

(Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)

Wash the sample with PBS for 3 times, 3 minutes each time.

Blocking

Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)

Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.

Immunofluorescence Staining (Day 1)

1. Remove the blocking solution and add the diluted primary antibody.

2. Incubate the sample in a humidified chamber at 4°C overnight.

Immunofluorescence Staining (Day 2)

1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.

2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.

3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.

4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.

5. Wash with PBST for 3 times, 5 minutes each time.

Mounting

1. Mount the sample with an anti-fade mounting medium.

2. Allow the slide to dry at room temperature overnight in the dark.

3. Store the slide in a slide storage box at 4°C, protected from light.

Datasheet & SDS

Biological Description

Specificity
Phospho-Vimentin (Ser56) Antibody (Rabbit mAb) [M10H3] detects endogenous levels of total Vimentin protein only when it is phosphorylated at Ser56.

Subcellular Location
Cell projection, Cytoplasm, Cytoskeleton, Intermediate filament, Membrane, Nucleus

Uniprot ID
P08670

Clone
M10H3

Synonym(s)
epididymis secretory sperm binding protein; FLJ36605; VIM; VIME; Vimentin

Background
Phosphorylation of Sin1 at threonine 86 (Thr86) represents a critical regulatory modification controlling the mechanistic target of rapamycin complex 2 (mTORC2), which comprises mTOR, rictor, Sin1, and mLST8 and functions as a key regulator of cell survival, growth, and cytoskeletal organization through phosphorylation of AGC kinase family members, including Akt at serine 473. Sin1 serves as an essential scaffolding component of mTORC2, and phosphorylation at Thr86 alongside Thr398 governs the integrity and catalytic activity of the entire mTORC2 complex through conformational changes that influence Sin1 association with other complex subunits. The functional consequences of Thr86 phosphorylation remain subject to cellular context, with two distinct regulatory paradigms emerging—Akt functions as the predominant kinase phosphorylating Sin1 at Thr86 across diverse cell lines and stimulation conditions, establishing a positive feedback loop wherein PDK1-mediated Akt phosphorylation at Thr308 generates partially active Akt that subsequently phosphorylates Sin1 at Thr86, enhancing mTORC2 kinase activity and enabling full Akt activation through mTORC2-mediated phosphorylation at Ser473. Conversely, phosphorylation of Sin1 at Thr86 and Thr398 by either S6K downstream of mTORC1 or by Akt itself triggers negative regulation by inducing Sin1 dissociation from the mTORC2 complex, thereby suppressing mTORC2 kinase activity and inhibiting Akt phosphorylation at Ser473 in response to insulin, IGF-1, PDGF, and EGF stimulation, establishing a feedback inhibition mechanism distinct from canonical IRS-1 and Grb10-mediated pathways. This dual regulatory capacity positions Thr86 phosphorylation as a molecular switch balancing mTORC2 activation and suppression depending on upstream signaling intensity, nutrient availability, and growth factor context. The phosphorylation state of Sin1 at Thr86 modulates mTORC2-dependent phosphorylation of additional substrates, including SGK1 at Ser422 and PKCα at Ser657, extending regulatory control beyond Akt to influence ion transport, cell migration, and membrane trafficking processes. Sin1 Thr86 phosphorylation integrates signals from both mTORC1 and growth factor receptor pathways, coordinating cellular responses to nutrient status and mitogenic stimulation through reciprocal crosstalk between mTORC1-S6K and mTORC2-Akt signaling axes. Cancer-associated mutations proximal to the Thr86 phosphorylation site, exemplified by the Sin1-R81T mutation identified from patient samples, impair phosphorylation-dependent negative regulation by preventing efficient phosphorylation, resulting in constitutive mTORC2 hyperactivation and sustained Akt signaling that drives oncogenic transformation, proliferation, and survival.

Background

Phosphorylation of Sin1 at threonine 86 (Thr86) represents a critical regulatory modification controlling the mechanistic target of rapamycin complex 2 (mTORC2), which comprises mTOR, rictor, Sin1, and mLST8 and functions as a key regulator of cell survival, growth, and cytoskeletal organization through phosphorylation of AGC kinase family members, including Akt at serine 473. Sin1 serves as an essential scaffolding component of mTORC2, and phosphorylation at Thr86 alongside Thr398 governs the integrity and catalytic activity of the entire mTORC2 complex through conformational changes that influence Sin1 association with other complex subunits. The functional consequences of Thr86 phosphorylation remain subject to cellular context, with two distinct regulatory paradigms emerging—Akt functions as the predominant kinase phosphorylating Sin1 at Thr86 across diverse cell lines and stimulation conditions, establishing a positive feedback loop wherein PDK1-mediated Akt phosphorylation at Thr308 generates partially active Akt that subsequently phosphorylates Sin1 at Thr86, enhancing mTORC2 kinase activity and enabling full Akt activation through mTORC2-mediated phosphorylation at Ser473. Conversely, phosphorylation of Sin1 at Thr86 and Thr398 by either S6K downstream of mTORC1 or by Akt itself triggers negative regulation by inducing Sin1 dissociation from the mTORC2 complex, thereby suppressing mTORC2 kinase activity and inhibiting Akt phosphorylation at Ser473 in response to insulin, IGF-1, PDGF, and EGF stimulation, establishing a feedback inhibition mechanism distinct from canonical IRS-1 and Grb10-mediated pathways. This dual regulatory capacity positions Thr86 phosphorylation as a molecular switch balancing mTORC2 activation and suppression depending on upstream signaling intensity, nutrient availability, and growth factor context. The phosphorylation state of Sin1 at Thr86 modulates mTORC2-dependent phosphorylation of additional substrates, including SGK1 at Ser422 and PKCα at Ser657, extending regulatory control beyond Akt to influence ion transport, cell migration, and membrane trafficking processes. Sin1 Thr86 phosphorylation integrates signals from both mTORC1 and growth factor receptor pathways, coordinating cellular responses to nutrient status and mitogenic stimulation through reciprocal crosstalk between mTORC1-S6K and mTORC2-Akt signaling axes. Cancer-associated mutations proximal to the Thr86 phosphorylation site, exemplified by the Sin1-R81T mutation identified from patient samples, impair phosphorylation-dependent negative regulation by preventing efficient phosphorylation, resulting in constitutive mTORC2 hyperactivation and sustained Akt signaling that drives oncogenic transformation, proliferation, and survival.

References
https://pubmed.ncbi.nlm.nih.gov/16990256/ https://pubmed.ncbi.nlm.nih.gov/19291697/

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%

Phospho-Vimentin (Ser56) Antibody (Rabbit mAb) [M10H3]

Usage Information

Experimental Methods

Datasheet & SDS

Biological Description

Tech Support