701068

antibody from Invitrogen Antibodies

Targeting: PRKAA2 AMPK, AMPKa2, PRKAA

Antibody data

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Validation data

Product number: 701068 - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-AMPK alpha-1,2 (Thr183, Thr172) Recombinant Rabbit Monoclonal Antibody (10H2L20)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.
Antibody clone number: 10H2L20
Concentration: 0.5 mg/mL

PRKAA2 protein structure - 701068 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with Metformin (10mM for 24 h) (Lane 2), Hep G2 (Lane 3), Hep G2 treated with Metformin (10mM for 24 h) (Lane 4), U-87 MG (Lane 5), and U-87 MG treated with AICAR (1mM for 48 h) (Lane 6). The blot was probed with Phospho-AMPK alpha-1,2 (Thr183, Thr172) Monoclonal Antibody (Product # 701068, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 63 kDa band corresponding to Phospho-AMPK alpha-1,2 (Thr183, Thr172) was observed across the cell lines tested and enhanced upon treatment.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with Metformin (10mM for 24 h) (Lane 2), Hep G2 (Lane 3), Hep G2 treated with Metformin (10mM for 24 h) (Lane 4), U-87 MG (Lane 5), and U-87 MG treated with AICAR (1mM for 48 h) (Lane 6). The blot was probed with Phospho-AMPK alpha-1,2 (Thr183, Thr172) Monoclonal Antibody (Product # 701068, 2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 63 kDa band corresponding to Phospho-AMPK alpha-1,2 (Thr183, Thr172) was observed across the cell lines tested and enhanced upon treatment.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of Phospho-AMPK alpha-1,2 (Thr183, Thr172) in whole cell extracts of HepG2 cells using a Phospho-AMPK alpha-1,2 (Thr183, Thr172) recombinant rabbit monoclonal antibody (Product # 701068) at a dilution of 2.5 µg/mL. To confirm specificity, competition was performed by preincubation with the phosphopeptide to inhibit antibody binding (lane 2). Results show a band at ~62kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of AMPKA (pThr183, Thr172) was done on 70% confluent log phase MDA-MB-231 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with Phospho-AMPK alpha-1,2 (Thr183, Thr172) (10H2L20) Recombinant Rabbit Monoclonal Antibody (Product # 701068) at 1 µg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing Nuclear localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-AMPK alpha-1,2 (Thr183, Thr172) was performed using 90% confluent log phase A-431 cells treated with 1mmol/mL Metformin for 24 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Phospho-AMPK alpha-1,2 (Thr183, Thr172) Recombinant Rabbit Monoclonal Antibody (Product # 701068) at 5µg/mL in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing nuclear localization. Panel e shows untreated cells with reduced nuclear signal. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.