TREM2-dependent mTOR Metabolic Fitness Antibody Sampler Kit
cat.: HAK21193
Product Type: Antibody Sampler Kit
Species reactivity: Human, Mouse
Applications: WB
Form: Liquid
Storage condition: Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.
Storage buffer: 1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration: 1ug/ul
Uniprot #: SwissProt: Q9NZC2 Human | Q99NH8 Mouse | Q13131 Human | Q5EG47 Mouse | P54645 Rat | Q13131 Human | P54646 Human | Q5EG47 Mouse | Q8BRK8 Mouse | P54645 Rat | Q09137 Rat | P42345 Human | Q9JLN9 Mouse | P42346 Rat | P42345 Human | Q9JLN9 Mouse | P31749 Human | P31751 Human | Q9Y243 Human | P31750 Mouse | Q60823 Mouse | Q9WUA6 Mouse | P47196 Rat | P47197 Rat | Q63484 Rat | P31749 Human | P31751 Human | Q9Y243 Human | P31750 Mouse | Q60823 Mouse | Q9WUA6 Mouse | P47196 Rat | P47197 Rat | Q63484 Rat | Q9H492 Human | Q91VR7 Mouse | Q6XVN8 Rat | Q9GZQ8 Human | Q9CQV6 Mouse | Q62625 Rat
Images
HAK21193_1.jpg Fig1: Western blot analysis of AMPK alpha 1 on different lysates with Rabbit anti-AMPK alpha 1 antibody (ET1608-40) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: MCF7 cell lysate (20 µg/Lane)
Lane 3: K-562 cell lysate (20 µg/Lane)
Lane 4: 293T cell lysate (20 µg/Lane)
Lane 5: HT-29 cell lysate (20 µg/Lane)
Lane 6: L-929 cell lysate (20 µg/Lane)
Lane 7: C6 cell lysate (20 µg/Lane)

Predicted band size: 64 kDa
Observed band size: 64 kDa

Exposure time: 1 minute 10 seconds; ECL: K1802;
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 (ET1608-40) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution were 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.
HAK21193_2.jpg Fig2: Immunocytochemistry analysis of HeLa cells labeling AMPK alpha 1 with Rabbit anti-AMPK alpha 1 antibody (ET1608-40) at 1/250 dilution and competitor's antibody at 1/250 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-AMPK alpha 1 antibody (ET1608-40) at 1/250 dilution and competitor's antibody at 1/250 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 (M1305-2, 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.
HAK21193_3.jpg Fig3: Western blot analysis of Phospho-AMPK alpha 1 (T183) +AMPK alpha 2 (T172) on different lysates with Rabbit anti-Phospho-AMPK alpha 1 (T183) +AMPK alpha 2 (T172) antibody (HA723603) at 1/2,000 dilution.

Lane 1: NCI-H1299 cell lysate
Lane 2: NCI-H1299 treated with 0.5μM Oligomycin for 15 minutes cell lysate
Lane 3: SH-SY5Y cell lysate
Lane 4: SH-SY5Y treated with 0.5μM Oligomycin for 30 minutes cell lysate
Lane 5: C2C12 cell lysate
Lane 6: C2C12 treated with 1mM AICAR for 30 minutes cell lysate
Lane 7: C6 cell lysate
Lane 8: C6 treated with 1μM Oligomycin for 30 minutes cell lysate
Lane 9: NCI-H1299 treated with 0.5μM Oligomycin for 15 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.
Predicted band size: 64 kDa
Observed band size: 64 kDa

Exposure time: 59 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 (HA723603) 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.
HAK21193_4.jpg Fig4: Immunohistochemical analysis of paraffin-embedded human bain tissue untreated / treated with λpp with Rabbit anti-Phospho-AMPK alpha 1 (T183) +AMPK alpha 2 (T172) antibody (HA723603) at 1/200 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 (HA723603) at 1/200 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.
HAK21193_5.jpg Fig5: Western blot analysis of mTOR on different lysates with Rabbit anti-mTOR antibody (ET1608-5) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: MCF7 cell lysate (20 µg/Lane)
Lane 3: HEK-293 cell lysate (20 µg/Lane)
Lane 4: HepG2 cell lysate (20 µg/Lane)
Lane 5: C2C12 cell lysate (20 µg/Lane)
Lane 6: PC-12 cell lysate (20 µg/Lane)
Lane 7: C6 cell lysate (20 µg/Lane)
Lane 8: Mouse testis tissue lysate (20 µg/Lane)
Lane 9: Rat testis tissue lysate (20 µg/Lane)

Predicted band size: 289 kDa
Observed band size: 289 kDa

Exposure time: 24 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 (ET1608-5) at 1/5,000 dilution and competitor's antibody at 1/1,000 dilution were 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.
HAK21193_6.jpg Fig6: Application: IF-Tissue

Species: Mouse

Site: testis

Sample: Paraffin-embedded section

Antibody concentration: 1/500
HAK21193_7.jpg Fig7: Western blot analysis of AKT1/2/3 on different lysates with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: MCF7 cell lysate
Lane 3: A549 cell lysate
Lane 4: U-2 OS cell lysate
Lane 5: NIH/3T3 cell lysate

Lysates/proteins at 15 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 30 seconds; ECL: K1802;

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 (HA721870) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were 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.
HAK21193_8.jpg Fig8: Immunocytochemistry analysis of MCF7 cells labeling AKT1/2/3 with Rabbit anti-AKT1/2/3 antibody (HA721870) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 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-AKT1/2/3 antibody (HA721870) at 1/100 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 (M1305-2, 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.
HAK21193_9.jpg Fig9: Western blot analysis of Phospho-AKT (S473) on different lysates with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/2,000 dilution and pan AKT antibody (HA721870) at 1/2,000 dilution.

Lane 1: MCF7 cell lysate
Lane 2: MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate
Lane 3: SH-SY5Y cell lysate
Lane 4: SH-SY5Y treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 5: HEK-293 cell lysate
Lane 6: HEK-293 treated with 50μM LY294002 for 6 hours cell lysate
Lane 7: NIH/3T3 cell lysate
Lane 8: NIH/3T3 treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 9: C6 cell lysate
Lane 10: C6 treated with 100ng/mL PDGF for 5 minutes cell lysate
Lane 11: MCF7 treated with 50ng/mL Calyculin A for 45 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 56 kDa
Observed band size: 56 kDa

Exposure time: 20 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% BSA for 1 hour at room temperature. The primary antibody (HA722129) at 1/2,000 dilution and pan AKT antibody (HA721870) at 1/2,000 dilution were used in 5% BSA at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
HAK21193_10.jpg Fig10: Immunohistochemical analysis of paraffin-embedded mouse spleen tissue untreated / treated with λpp / phospho-peptide / non-phospho-peptide with Rabbit anti-Phospho-AKT (S473) antibody (HA722129) at 1/200 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (HA722129) at 1/200 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.
HAK21193_11.jpg Fig11: Flow cytometric analysis of NIH/3T3 cells untreated (left) / treated with 100ng/mL PDGF for 1 hour (right) labeling Phospho-AKT (S473).

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722129, 0.1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
HAK21193_12.jpg Fig12: Western blot analysis of MAP1LC3A on different lysates with Rabbit anti-MAP1LC3A antibody (ET1609-26) at 1/1,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate
Lane 3: C2C12 cell lysate
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate
Lane 5: C6 cell lysate
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 14 kDa
Observed band size: 14/16 kDa

Exposure time: 3 minutes; 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 (ET1609-26) at 1/1,000 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.
HAK21193_13.jpg Fig13: Immunocytochemistry analysis of C6 cells labeling MAP1LC3A with Rabbit anti-MAP1LC3A antibody (ET1609-26) at 1/100 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-MAP1LC3A antibody (ET1609-26) at 1/100 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.
HAK21193_14.jpg Fig14: Western blot analysis of LC3B on different lysates with Rabbit anti-LC3B antibody (ET1701-65) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution.

Lane 1: HeLa cell lysate (20 µg/Lane)
Lane 2: HeLa treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 3: C2C12 cell lysate (20 µg/Lane)
Lane 4: C2C12 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 5: C6 cell lysate (20 µg/Lane)
Lane 6: C6 treated with 50μM Chloroquine for 18 hours cell lysate (20 µg/Lane)
Lane 7: mouse brain tissue lysate (20 µg/Lane)
Lane 8: rat brain tissue lysate (20 µg/Lane)

Predicted band size: 14/16 kDa
Observed band size: 14/16 kDa

Exposure time: 3 minutes;
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 (ET1701-65) at 1/2,000 dilution and competitor's antibody at 1/1,000 dilution were 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.
HAK21193_15.png Fig15: Immunocytochemistry analysis of HeLa cells treated with or without 50μM Chloroquine for 24 hours labeling LC3B with Rabbit anti-LC3B antibody (ET1701-65) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-LC3B antibody (ET1701-65) at 1/200 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, 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.
HAK21193_16.jpg Fig16: Fluorescence multiplex immunohistochemical analysis of human gastric cancer (Formalin/PFA-fixed paraffin-embedded sections). Panel A: the merged image of anti-LC3B (ET1701-65, Green), anti-CD31 (M1511-8, Red) and anti-CA-IX (ET1701-51, Yellow) on human gastric cancer. HRP Conjugated UltraPolymer Goat Polyclonal Antibody HA1119/HA1120 was used as a secondary antibody. The immunostaining was performed with the Sequential Immuno-staining Kit (IRISKitCmTSA Kit 900802). The section was incubated in three rounds of staining: in the order of ET1701-65 (1/100 dilution), M1511-8 (1/2,000 dilution) and ET1701-51 (1/100 dilution) for 20 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 30 mins at 95℃. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Olympus VS200 Slide Scanner.
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