Phospho-AS160 (T642) Recombinant Rabbit Monoclonal Antibody [JE60-97]
cat.: HA721489
Product Type: Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity: Human, Mouse, Rat
Applications: WB, IF-Cell
Clonality: Monoclonal
Clone number: JE60-97
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
Purification: Protein A affinity purified.
Molecular weight: Predicted band size: 147 kDa
Isotype: IgG
Immunogen: Synthetic phosphopeptide corresponding to residues surrounding Thr642 of human AS160 protein.
Positive control: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, NIH/3T3 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, PC-12 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, NIH/3T3 cells starved overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes, 293T cells starved overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes.
Subcellular location: Cytoplasm.
Recommended Dilutions:
  WB
  IF-Cell
1:1,000
1:100
Uniprot #: SwissProt: O60343 Human | Q8BYJ6 Mouse
Entrez Gene: 306117 Rat
Alternative names: Acrg embryonic lethality (mouse) minimal region ortholog Acrg embryonic lethality minimal region ortholog Acrg embryonic lethality mouse minimal region ortholog Akt substrate of 160 kDa AS 160 AS160 BUB2 CDC16 KIAA0603 NIDDM5 TBC (Tre 2 BUB2 CDC16) domain containing protein TBC Tre 2 BUB2 CDC16 domain containing protein TBC1 D4 TBC1 domain family member 4 Tbc1d4 TBCD4_HUMAN Tre-2
Images
HA721489_1.jpg Fig1: Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/1,000 dilution.

Lane 1: NIH/3T3 cell lysate
Lane 2: NIH/3T3 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 147 kDa
Observed band size: 160 kDa

Exposure time: 25 seconds;

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 (HA721489) 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.
HA721489_2.jpg Fig2: Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/1,000 dilution.

Lane 1: PC-12 cell lysate
Lane 2: PC-12 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 147 kDa
Observed band size: 160 kDa

Exposure time: 25 seconds;

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 (HA721489) 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.
HA721489_3.jpg Fig3: Western blot analysis of Phospho-AS160 (T642) on different lysates with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) at 1/1,000 dilution.

Lane 1: HEK-293 cell lysate
Lane 2: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate
Lane 3: HEK-293 cell lysate, the membrane treated with λpp for 1 hour
Lane 4: HEK-293 starve overnight then treated with 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes cell lysate, then the membrane treated with λpp for 1 hour

Lysates/proteins at 20 µg/Lane.

Predicted band size: 147 kDa
Observed band size: 160 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 (HA721489) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature.
HA721489_4.jpg Fig4: Immunocytochemistry analysis of NIH/3T3 cells starved overnight then treated with or without 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes labeling Phospho-AS160 (T642) with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) 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-Phospho-AS160 (T642) antibody (HA721489) 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.
HA721489_5.jpg Fig5: Immunocytochemistry analysis of 293T cells starved overnight then treated with or without 100nM Calyculin A for 50 minutes add 100ng/mL insulin for 20 minutes labeling Phospho-AS160 (T642) with Rabbit anti-Phospho-AS160 (T642) antibody (HA721489) 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-Phospho-AS160 (T642) antibody (HA721489) 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.
Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.