Phospho-GCN2 (T899) Recombinant Rabbit Monoclonal Antibody [PSH10-57]
cat.: HA723221
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell, IHC-P
Clonality: Monoclonal
Clone number: PSH10-57
Form: Liquid
Storage condition: Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.
Storage buffer: PBS (pH7.4), 0.1% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 187 kDa
Isotype: IgG
Immunogen: Synthetic phosphopeptide corresponding to residues surrounding Thr899 of Human GCN2.
Positive control: HeLa cell lysate, HeLa starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate, Neuro-2a cell lysate, Neuro-2a treated with 100nM Calyculin A for 30 minutes cell lysate, C6 cell lysate, C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate, HeLa cells treated with 100nM Calyculin A for 30 minutes, Neuro-2a cells treated with 100nM Calyculin A for 30 minutes, C6 cells treated with 100ng/mL Calyculin A for 1 hour, human brain tissue, mouse brain tissue, rat brain tissue.
Subcellular location: Cytoplasm.
Recommended Dilutions:
  WB
  IF-Cell
  IHC-P
1:2,000-1:5,000
1:5,000
1:200-1:1,000
Uniprot #: SwissProt: Q9P2K8 Human | Q9QZ05 Mouse
Entrez Gene: 114859 Rat
Alternative names: E2AK4_HUMAN Eif2ak4 Eukaryotic Translation Initiation Factor 2 alpha kinase 4 Eukaryotic translation initiation factor 2-alpha kinase 4 GCN2 GCN2 eIF2alpha kinase GCN2 like protein GCN2-like protein KIAA1338 MGCN2
Images
HA723221_1.jpg Fig1: Western blot analysis of Phospho-GCN2 (T899) on different lysates with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/2,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)
Lane 3: Neuro-2a cell lysate (20 µg/Lane)
Lane 4: Neuro-2a treated with 100nM Calyculin A for 30 minutes cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate (20 µg/Lane)
Lane 7: HeLa starved for 3 hours then treated with 100nM Calyculin A for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour (20 µg/Lane)
Lane 8: Neuro-2a treated with 100nM Calyculin A for 30 minutes cell lysate, then the membrane treated with λpp for 1 hour (20 µg/Lane)
Lane 9: C6 treated with 100ng/mL Calyculin A for 1 hour cell lysate, then the membrane treated with λpp for 1 hour (20 µg/Lane)

Predicted band size: 187 kDa
Observed band size: 250+ kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA723221) at 1/2,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HA723221_2.jpg Fig2: Immunocytochemistry analysis of HeLa cells untreated / treated with 100nM Calyculin A for 30 minutes labeling Phospho-GCN2 (T899) with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/5,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/5,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA723221_3.jpg Fig3: Immunocytochemistry analysis of Neuro-2a cells untreated / treated with 100nM Calyculin A for 30 minutes labeling Phospho-GCN2 (T899) with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/5,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/5,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA723221_4.jpg Fig4: Immunocytochemistry analysis of C6 cells untreated / treated with 100ng/mL Calyculin A for 1 hour labeling Phospho-GCN2 (T899) with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/5,000 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/5,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
HA723221_5.jpg Fig5: Immunohistochemical analysis of paraffin-embedded human brain tissue untreated / treated with λpp with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723221) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HA723221_6.jpg Fig6: Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723221) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
HA723221_7.jpg Fig7: Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Phospho-GCN2 (T899) antibody (HA723221) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA723221) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
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