SMRT Antibody [M7D13] | Primary Antibodies

Application: Reactivity: Human, Monkey

Lane 1: MOLT-4

Experiment Essentials

WB
Recommended SDS-PAGE separating gel concentration: 5%.
Recommended wet transfer conditions: 250 mA, 180 min.

Usage Information

Dilution
WB1:1000 CHIP1:50

Application
WB, ChIP

Reactivity
Human, 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
270 kDa

Positive Control	MOLT-4 cells; SW620 cells; HeLa cells; 293T cells; LS-180 cells (Calcitriol, 10 nM, 3 h)
Negative Control

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/Nuclear 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/Nuclear 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/Nuclear 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: 250 mA, 180 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/Nuclear 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/Nuclear 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/Nuclear 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: 250 mA, 180 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
SMRT Antibody [M7D13] detects endogenous levels of total SMRT protein.

Subcellular Location
Nucleus

Uniprot ID
Q9Y618

Clone
M7D13

Synonym(s)
Silencing mediator of retinoic acid and thyroid hormone receptor; SMRT; NCOR2

Background
SMRT belongs to the NCoR/SMRT family of nuclear receptor corepressors that mediate transcriptional silencing by assembling multi-subunit repressive complexes on unliganded nuclear receptors. SMRT organizes N-terminal repression domains interspersed with SANT motifs alongside C-terminal CoRNR box interaction domains that form amphipathic helices docking into hydrophobic grooves on receptor ligand-binding domains. SMRT bridges nuclear receptors to HDAC3 through a deacetylase activation domain encompassing the first SANT motif that allosterically relieves HDAC3 autoinhibition by stabilizing its catalytic pocket, while the second SANT motif forms a histone interaction domain that preferentially binds unacetylated H3/H4 tails to enhance substrate presentation and amplify deacetylation at target promoters. Repression domains recruit mSin3A, GPS2, and TBL1/TBLR1 scaffolds that propagate silencing through coordinated histone hypoacetylation, DNA methylation, and chromatin compaction via recruitment of HP1 and PRC2 complexes. Interaction with thyroid hormone and retinoic acid receptors enforces lineage-specific gene repression during development, with alternative splicing generating isoforms that modulate receptor specificity through variable usage of the CoRNR box. SMRT governs metabolic homeostasis by silencing lipogenic programs in liver and adipogenesis in mesenchymal precursors, with dynamic exchange dictated by ligand-induced conformational shifts that evict corepressor for coactivator binding. Dysregulation through translocation or mutation impairs hormone responsiveness in acute promyelocytic leukemia and thyroid disorders.

Background

SMRT belongs to the NCoR/SMRT family of nuclear receptor corepressors that mediate transcriptional silencing by assembling multi-subunit repressive complexes on unliganded nuclear receptors. SMRT organizes N-terminal repression domains interspersed with SANT motifs alongside C-terminal CoRNR box interaction domains that form amphipathic helices docking into hydrophobic grooves on receptor ligand-binding domains. SMRT bridges nuclear receptors to HDAC3 through a deacetylase activation domain encompassing the first SANT motif that allosterically relieves HDAC3 autoinhibition by stabilizing its catalytic pocket, while the second SANT motif forms a histone interaction domain that preferentially binds unacetylated H3/H4 tails to enhance substrate presentation and amplify deacetylation at target promoters. Repression domains recruit mSin3A, GPS2, and TBL1/TBLR1 scaffolds that propagate silencing through coordinated histone hypoacetylation, DNA methylation, and chromatin compaction via recruitment of HP1 and PRC2 complexes. Interaction with thyroid hormone and retinoic acid receptors enforces lineage-specific gene repression during development, with alternative splicing generating isoforms that modulate receptor specificity through variable usage of the CoRNR box. SMRT governs metabolic homeostasis by silencing lipogenic programs in liver and adipogenesis in mesenchymal precursors, with dynamic exchange dictated by ligand-induced conformational shifts that evict corepressor for coactivator binding. Dysregulation through translocation or mutation impairs hormone responsiveness in acute promyelocytic leukemia and thyroid disorders.

References
https://pubmed.ncbi.nlm.nih.gov/12840002/ https://pubmed.ncbi.nlm.nih.gov/9415406/

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%