GCN1 Antibody [G16A4] | Primary Antibodies

Application: Reactivity: Human, Mouse, Rat

Lane 1: Hela

Experiment Essentials

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

Usage Information

Dilution
WB1:1000

Application
WB

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 Observed MW
293 kDa 295 kDa,124 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.

Positive Control	Human skeletal muscle tissue; Mouse brain tissue; Mouse liver tissue; Rat liver tissue; HEK293T cells; PC-3 cells; DU-145 cells; HeLa 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 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) , 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) , 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: 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 primary antibody dilution buffer 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 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) , 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) , 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: 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 primary antibody dilution buffer 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
GCN1 Antibody (Rabbit mAb) [G16A4] detects endogenous levels of total GCN1 protein.

Subcellular Location
Cytoplasm

Uniprot ID
Q92616

Clone
G16A4

Synonym(s)
GCN1L1 \| KIAA0219 \| GCN1 \| Stalled ribosome sensor GCN1 \| GCN1 eIF-2-alpha kinase activator homolog \| GCN1-like protein 1 \| General control of amino-acid synthesis 1-like protein 1 \| Translational activator GCN1 \| HsGCN1

Background
GCN1 (GCN1 activator of EIF2AK4) is a large ribosome‑bound regulatory protein that functions as a collision sensor and upstream activator of the protein kinase GCN2, thereby integrating translational status and amino acid availability with the integrated stress response and co‑translational quality control. The protein contains extended HEAT‑like repeats and an EF3‑related region that contacts elongating ribosomes and forms a complex with its partner GCN20; this architecture allows GCN1 to bind disomes and higher‑order collided ribosome assemblies and to position GCN2 at the interface between ribosomal subunits and the A‑site region. During amino acid limitation or other stresses that slow elongation, ribosomes stall with unoccupied A sites, trailing ribosomes collide with the stalled ones, and GCN1 is recruited to these collided ribosomes, where it in turn recruits and stabilizes GCN2, enabling GCN2 activation by specific interactions with the collided ribosome and by local enrichment of uncharged tRNA cognate to the A‑site codon. Activated GCN2 phosphorylates the α subunit of eukaryotic initiation factor 2, which converts eIF2 into an inhibitor of ternary complex recycling, attenuates global cap‑dependent translation, and promotes selective translation of stress‑responsive mRNAs such as ATF4, thereby triggering a transcriptional program that upregulates amino acid transporters and biosynthetic enzymes and restores metabolic balance. Beyond this canonical amino acid response, GCN1 contributes to responses to UV irradiation and other stresses that induce ribosome pausing, and mammalian genetic analyses show that GCN1 is essential for embryonic development, cell proliferation, and proper G2/M progression, linking its ribosome‑associated functions to cell‑cycle regulation. GCN1 also participates directly in ribosome‑centric quality control: GCN1 binding to collided ribosomes promotes engagement of the RNF14–RNF25 ubiquitin ligase pathway, which targets elongation factor eEF1A and release factor eRF1 for ubiquitination and degradation and modifies ribosomal proteins, coupling persistent stalling to the clearance of key translation factors and reconfiguration of the translational machinery.

Background

GCN1 (GCN1 activator of EIF2AK4) is a large ribosome‑bound regulatory protein that functions as a collision sensor and upstream activator of the protein kinase GCN2, thereby integrating translational status and amino acid availability with the integrated stress response and co‑translational quality control. The protein contains extended HEAT‑like repeats and an EF3‑related region that contacts elongating ribosomes and forms a complex with its partner GCN20; this architecture allows GCN1 to bind disomes and higher‑order collided ribosome assemblies and to position GCN2 at the interface between ribosomal subunits and the A‑site region. During amino acid limitation or other stresses that slow elongation, ribosomes stall with unoccupied A sites, trailing ribosomes collide with the stalled ones, and GCN1 is recruited to these collided ribosomes, where it in turn recruits and stabilizes GCN2, enabling GCN2 activation by specific interactions with the collided ribosome and by local enrichment of uncharged tRNA cognate to the A‑site codon. Activated GCN2 phosphorylates the α subunit of eukaryotic initiation factor 2, which converts eIF2 into an inhibitor of ternary complex recycling, attenuates global cap‑dependent translation, and promotes selective translation of stress‑responsive mRNAs such as ATF4, thereby triggering a transcriptional program that upregulates amino acid transporters and biosynthetic enzymes and restores metabolic balance. Beyond this canonical amino acid response, GCN1 contributes to responses to UV irradiation and other stresses that induce ribosome pausing, and mammalian genetic analyses show that GCN1 is essential for embryonic development, cell proliferation, and proper G2/M progression, linking its ribosome‑associated functions to cell‑cycle regulation. GCN1 also participates directly in ribosome‑centric quality control: GCN1 binding to collided ribosomes promotes engagement of the RNF14–RNF25 ubiquitin ligase pathway, which targets elongation factor eEF1A and release factor eRF1 for ubiquitination and degradation and modifies ribosomal proteins, coupling persistent stalling to the clearance of key translation factors and reconfiguration of the translational machinery.

References
https://pubmed.ncbi.nlm.nih.gov/38474243/ https://pubmed.ncbi.nlm.nih.gov/41037622/

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%

GCN1 Antibody (Rabbit mAb) [G16A4]