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Anti-Phospho-TFEB (Ser211) Rabbit Antibody [N21G19]

Catalog No.: F1474

Application: Reactivity: Human

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human

Source
Rabbit

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
70 kDa

Positive Control	Raji cell; Daudi cell; HDLM-2 cell
Negative Control

Exprimental 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. 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) and put the sample on ice for 5 min. 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) and put the sample on ice for 5 min. 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 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 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.

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.

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) and put the sample on ice for 5 min.

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) and put the sample on ice for 5 min.

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 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

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-TFEB (Ser211) Rabbit mAb recognizes endogenous levels of TFEB protein only when phosphorylated at Ser211.

Subcellular Location
Cytoplasm, Lysosome, Membrane, Nucleus

Uniprot ID
P19484

Clone
N21G19

Synonym(s)
Transcription factor EB , Class E basic helix-loop-helix protein 35 (bHLHe35), TFEB, BHLHE35

Background
Phospho-TFEB (Ser211) refers to the phosphorylated form of transcription factor EB (TFEB) at serine residue 211, a critical regulatory site within its structure. TFEB is a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor of the MiT/TFE family that controls genes involved in autophagy, lysosomal biogenesis, and cellular clearance. The full-length TFEB protein contains DNA-binding, dimerization, and transactivation domains, with Ser211 located in a region that regulates subcellular localization. Structurally defined as having Gln glutamine-rich region, AD acidic domain, bHLH basic helix–loop–helix, LZ leucine zipper domain, Pro proline-rich segment. Under nutrient-rich conditions, mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates Ser211 at the lysosomal surface, creating a binding site for 14-3-3 chaperone proteins. This interaction masks TFEB’s nuclear localization signal (NLS), sequestering it in the cytoplasm and preventing transcriptional activation of its target genes. When cells undergo stress or starvation, mTORC1 activity decreases, leading to Ser211 dephosphorylation, release from 14-3-3, and nuclear translocation of TFEB. There, TFEB activates genes containing the CLEAR motif, enhancing autophagy and lysosomal function. Thus, phosphorylation of Ser211 is essential for dynamically regulating TFEB’s activity in response to cellular metabolic states, maintaining energy homeostasis and facilitating clearance of toxic substrates in diseases such as neurodegeneration and kidney disorders.

Background

Phospho-TFEB (Ser211) refers to the phosphorylated form of transcription factor EB (TFEB) at serine residue 211, a critical regulatory site within its structure. TFEB is a basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factor of the MiT/TFE family that controls genes involved in autophagy, lysosomal biogenesis, and cellular clearance. The full-length TFEB protein contains DNA-binding, dimerization, and transactivation domains, with Ser211 located in a region that regulates subcellular localization. Structurally defined as having Gln glutamine-rich region, AD acidic domain, bHLH basic helix–loop–helix, LZ leucine zipper domain, Pro proline-rich segment. Under nutrient-rich conditions, mechanistic target of rapamycin complex 1 (mTORC1) phosphorylates Ser211 at the lysosomal surface, creating a binding site for 14-3-3 chaperone proteins. This interaction masks TFEB’s nuclear localization signal (NLS), sequestering it in the cytoplasm and preventing transcriptional activation of its target genes. When cells undergo stress or starvation, mTORC1 activity decreases, leading to Ser211 dephosphorylation, release from 14-3-3, and nuclear translocation of TFEB. There, TFEB activates genes containing the CLEAR motif, enhancing autophagy and lysosomal function. Thus, phosphorylation of Ser211 is essential for dynamically regulating TFEB’s activity in response to cellular metabolic states, maintaining energy homeostasis and facilitating clearance of toxic substrates in diseases such as neurodegeneration and kidney disorders.

References
https://pubmed.ncbi.nlm.nih.gov/32377395/ https://pubmed.ncbi.nlm.nih.gov/27252382/

Reference Table for Selecting PVDF Membrane Pore Size Specifications

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

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3
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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%