SMG-1 Antibody [L4N6] | Primary Antibodies

Application: Reactivity: Human, Monkey

Lane 1: U2OS, Lane 2: 293, Lane 3: MCF7

Experiment Essentials

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

Usage Information

Dilution
WB1:1000 IP1:50

Application
WB, IP

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
410 kDa

Positive Control	U-2 OS cells; 293 cells; MCF7 cells
Negative Control

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)，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: 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/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: 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
SMG-1 Antibody [L4N6] detects endogenous levels of total SMG-1 protein.

Subcellular Location
Cytoplasm, Nucleus

Uniprot ID
Q96Q15

Clone
L4N6

Synonym(s)
Serine/threonine-protein kinase SMG1; SMG-1; hSMG-1; Lambda/iota protein kinase C-interacting protein; Lambda-interacting protein; Nonsense mediated mRNA decay-associated PI3K-related kinase SMG1; SMG1; ATX; KIAA0421; LIP

Background
SMG‑1 is a large serine/threonine kinase and a member of the phosphoinositide 3‑kinase‑related kinase (PIKK) family, which includes ATM, ATR, mTOR, DNA‑PKcs, and TRRAP, and it is widely expressed in mammalian cells where it functions as a core regulator of mRNA quality control and stress‑responsive signaling. SMG‑1 forms a stable complex with SMG‑8 and SMG‑9, and this assembly targets the RNA‑binding protein UPF1 (hUpf1) for phosphorylation following recognition of premature termination codons on spliced mRNAs, thereby triggering nonsense‑mediated mRNA decay (NMD) and preventing the accumulation of truncated, nonfunctional or aberrant proteins. SMG‑1 phosphorylates p53 and other substrates in response to DNA damage and genotoxic stress, and it contributes to cell‑cycle control by modulating the G1/S checkpoint through both p53‑dependent and p53‑independent mechanisms, including regulation of Cdc25A and suppression of CDK2 activity. SMG‑1 activity intersects with DNA‑damage and survival pathways, and exhibits the potential to protect cells from extrinsically induced apoptosis by influencing cell‑death‑receptor signaling and by stabilizing anti‑apoptotic regulators such as c‑FLIP, which dampens caspase‑8 activation in response to TNFα and Smac‑mimetic compounds. Loss or inhibition of SMG‑1 increases sensitivity to DNA‑damaging agents and to extrinsic apoptotic stimuli, and reduced SMG‑1 expression or function has been associated with impaired NMD, altered cell‑cycle progression, and either tumor‑suppressive or context‑dependent oncogenic phenotypes in different cancers.

Background

SMG‑1 is a large serine/threonine kinase and a member of the phosphoinositide 3‑kinase‑related kinase (PIKK) family, which includes ATM, ATR, mTOR, DNA‑PKcs, and TRRAP, and it is widely expressed in mammalian cells where it functions as a core regulator of mRNA quality control and stress‑responsive signaling. SMG‑1 forms a stable complex with SMG‑8 and SMG‑9, and this assembly targets the RNA‑binding protein UPF1 (hUpf1) for phosphorylation following recognition of premature termination codons on spliced mRNAs, thereby triggering nonsense‑mediated mRNA decay (NMD) and preventing the accumulation of truncated, nonfunctional or aberrant proteins. SMG‑1 phosphorylates p53 and other substrates in response to DNA damage and genotoxic stress, and it contributes to cell‑cycle control by modulating the G1/S checkpoint through both p53‑dependent and p53‑independent mechanisms, including regulation of Cdc25A and suppression of CDK2 activity. SMG‑1 activity intersects with DNA‑damage and survival pathways, and exhibits the potential to protect cells from extrinsically induced apoptosis by influencing cell‑death‑receptor signaling and by stabilizing anti‑apoptotic regulators such as c‑FLIP, which dampens caspase‑8 activation in response to TNFα and Smac‑mimetic compounds. Loss or inhibition of SMG‑1 increases sensitivity to DNA‑damaging agents and to extrinsic apoptotic stimuli, and reduced SMG‑1 expression or function has been associated with impaired NMD, altered cell‑cycle progression, and either tumor‑suppressive or context‑dependent oncogenic phenotypes in different cancers.

References
https://pubmed.ncbi.nlm.nih.gov/16289965/ https://pubmed.ncbi.nlm.nih.gov/24107632/

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%