Phospho-Twist (Ser68) Antibody [M15K16]

Application: Reactivity: Human

Lane 1: HEK-293T, Lane 2: HEK-293T (TWIST1(human)-3×Myc transfected), Lane 3: HEK-293T (TWIST1(human)-3×Myc transfected; alkaline phosphatase, 1h)

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:5000 IP1:60

Application
WB, IP

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 Observed MW
21 kDa 47 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.

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, 60 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 1:5000), 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, 60 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 1:5000), 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-Twist (Ser68) Antibody (Rabbit mAb) [M15K16] detects endogenous levels of total Twist protein only when it is phosphorylated at Ser68.

Subcellular Location
Nucleus

Uniprot ID
Q15672

Clone
M15K16

Synonym(s)
BHLHA38, TWIST, TWIST1, Twist-related protein 1, Class A basic helix-loop-helix protein 38, H-twist, bHLHa38

Background
Phospho‑Twist (Ser68) refers to Twist1 phosphorylated at serine 68, a basic helix–loop–helix transcription factor modification that marks a MAPK‑responsive, stabilized form of the protein linking upstream Ras–RTK–TGF‑β signaling to epithelial–mesenchymal transition, invasion, and drug resistance programs in cancer. Ser68 lies in the N‑terminal regulatory region outside the DNA‑binding basic helix–loop–helix core, and is a major phospho‑acceptor site for ERK1/2, JNK, and p38, which phosphorylate Twist1 at this residue when these kinases are activated by mitogens, oncogenic Ras, or TGF‑β. Phosphorylation at Ser68 reduces Twist1 ubiquitination and proteasomal degradation, increases protein half‑life, and raises steady‑state Twist1 levels without altering TWIST1 transcription, so the Ser68‑phosphorylated pool directly reflects MAPK activity and dictates how much Twist1 is available to form dimers and occupy E‑box–containing promoters and enhancers. Stabilized phospho‑Twist (Ser68) enhances repression of epithelial genes and induction of mesenchymal and invasion‑associated genes, thereby strengthening Twist1‑dependent epithelial–mesenchymal transition, increasing motility and invasiveness of breast cancer cells, and associating with resistance to paclitaxel and other microtubule‑targeting chemotherapy. Levels of Ser68‑phosphorylated Twist1 correlate with total Twist1 and activated JNK in invasive ductal breast carcinomas and are higher in progesterone receptor–negative and HER2‑positive tumors than in other subtypes, highlighting this phospho‑epitope as a readout of aggressive, MAPK‑driven Twist1 signaling in patient samples. Dephosphorylation is mediated by small C‑terminal domain phosphatase 1, which targets Ser(P)68‑Twist1, accelerates Twist1 degradation, and suppresses cell migration and invasion, defining a reversible kinase–phosphatase module that fine‑tunes Twist1 protein turnover and EMT output.

Background

Phospho‑Twist (Ser68) refers to Twist1 phosphorylated at serine 68, a basic helix–loop–helix transcription factor modification that marks a MAPK‑responsive, stabilized form of the protein linking upstream Ras–RTK–TGF‑β signaling to epithelial–mesenchymal transition, invasion, and drug resistance programs in cancer. Ser68 lies in the N‑terminal regulatory region outside the DNA‑binding basic helix–loop–helix core, and is a major phospho‑acceptor site for ERK1/2, JNK, and p38, which phosphorylate Twist1 at this residue when these kinases are activated by mitogens, oncogenic Ras, or TGF‑β. Phosphorylation at Ser68 reduces Twist1 ubiquitination and proteasomal degradation, increases protein half‑life, and raises steady‑state Twist1 levels without altering TWIST1 transcription, so the Ser68‑phosphorylated pool directly reflects MAPK activity and dictates how much Twist1 is available to form dimers and occupy E‑box–containing promoters and enhancers. Stabilized phospho‑Twist (Ser68) enhances repression of epithelial genes and induction of mesenchymal and invasion‑associated genes, thereby strengthening Twist1‑dependent epithelial–mesenchymal transition, increasing motility and invasiveness of breast cancer cells, and associating with resistance to paclitaxel and other microtubule‑targeting chemotherapy. Levels of Ser68‑phosphorylated Twist1 correlate with total Twist1 and activated JNK in invasive ductal breast carcinomas and are higher in progesterone receptor–negative and HER2‑positive tumors than in other subtypes, highlighting this phospho‑epitope as a readout of aggressive, MAPK‑driven Twist1 signaling in patient samples. Dephosphorylation is mediated by small C‑terminal domain phosphatase 1, which targets Ser(P)68‑Twist1, accelerates Twist1 degradation, and suppresses cell migration and invasion, defining a reversible kinase–phosphatase module that fine‑tunes Twist1 protein turnover and EMT output.

References
https://pubmed.ncbi.nlm.nih.gov/26975371/ https://pubmed.ncbi.nlm.nih.gov/21502402/

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-Twist (Ser68) Antibody (Rabbit mAb) [M15K16]