Hamartin/TSC1 Antibody [N1E19] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

Reactivity
Human, Mouse, Rat

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
150-170 kDa

Positive Control	MEF cells; HT-1080 cells; Rat2 cells; HAP1 cells
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)，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) 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) 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: 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 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.

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)，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) 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) 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: 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 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
Hamartin/TSC1 Antibody [N1E19] detects endogenous levels of total Hamartin/TSC1 protein.

Subcellular Location
Cytoplasm, Lysosome, Membrane

Uniprot ID
Q92574

Clone
N1E19

Synonym(s)
Hamartin; Tuberous sclerosis 1 protein; TSC1 1; KIAA0243; TSC

Background
Hamartin, the protein product of the TSC1 tumor suppressor gene, forms a heterodimeric GTPase-activating protein complex with tuberin (TSC2) that negatively regulates mTORC1 signaling. This complex accelerates GTP hydrolysis on Rheb, inhibiting downstream anabolic processes such as protein synthesis through S6K1 phosphorylation and 5'TOP mRNA translation, particularly under conditions of growth factor, nutrient, or energy stress. Hamartin contains an N-terminal coiled-coil domain that mediates heterodimerization with TSC2 via electrostatic interactions, a central Rho-activation domain with key arginine and lysine motifs that facilitate anchoring to the actin cytoskeleton through ERM proteins like ezrin, radixin, and moesin, and several phosphorylation sites targeted by CDK1 during the G2/M phase to modulate the stability of the complex. The TSC1-TSC2 complex integrates various signals from pathways such as AKT, AMPK, and REDD1 at the lysosome to suppress Rheb-GTP, thereby preventing recruitment and activation of mTORC1 and maintaining cellular quiescence. Independent of TSC2, hamartin also promotes focal adhesion assembly and RhoA activation, supporting cell adhesion, migration, neuronal intermediate filament anchoring, and vesicular trafficking. Hamartin is essential for regulating proliferation during organogenesis. Loss of TSC1 leads to constitutive mTORC1 hyperactivity, resulting in the formation of hamartomas in the brain (such as cortical tubers and subependymal giant cell astrocytomas), kidney (angiomyolipomas), heart (rhabdomyomas), and skin. Pathogenic mutations typically disrupt TSC2 binding or the coiled-coil domain stability, causing tuberous sclerosis complex, an autosomal dominant disorder characterized by epilepsy, autism, and tumor development.

Background

Hamartin, the protein product of the TSC1 tumor suppressor gene, forms a heterodimeric GTPase-activating protein complex with tuberin (TSC2) that negatively regulates mTORC1 signaling. This complex accelerates GTP hydrolysis on Rheb, inhibiting downstream anabolic processes such as protein synthesis through S6K1 phosphorylation and 5'TOP mRNA translation, particularly under conditions of growth factor, nutrient, or energy stress. Hamartin contains an N-terminal coiled-coil domain that mediates heterodimerization with TSC2 via electrostatic interactions, a central Rho-activation domain with key arginine and lysine motifs that facilitate anchoring to the actin cytoskeleton through ERM proteins like ezrin, radixin, and moesin, and several phosphorylation sites targeted by CDK1 during the G2/M phase to modulate the stability of the complex. The TSC1-TSC2 complex integrates various signals from pathways such as AKT, AMPK, and REDD1 at the lysosome to suppress Rheb-GTP, thereby preventing recruitment and activation of mTORC1 and maintaining cellular quiescence. Independent of TSC2, hamartin also promotes focal adhesion assembly and RhoA activation, supporting cell adhesion, migration, neuronal intermediate filament anchoring, and vesicular trafficking. Hamartin is essential for regulating proliferation during organogenesis. Loss of TSC1 leads to constitutive mTORC1 hyperactivity, resulting in the formation of hamartomas in the brain (such as cortical tubers and subependymal giant cell astrocytomas), kidney (angiomyolipomas), heart (rhabdomyomas), and skin. Pathogenic mutations typically disrupt TSC2 binding or the coiled-coil domain stability, causing tuberous sclerosis complex, an autosomal dominant disorder characterized by epilepsy, autism, and tumor development.

References
https://pubmed.ncbi.nlm.nih.gov/10806479/ https://pubmed.ncbi.nlm.nih.gov/23857276/

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

Handling Instructions

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

If you have any other enquiries, please leave a message.

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%