44-1150G

antibody from Invitrogen Antibodies

Targeting: PRKAA2 AMPK, AMPKa2, PRKAA

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Validation data

PRKAA2 protein structure - 44-1150G shown in red.

Richter RP, Ashtekar AR, Zheng L, Pretorius D, Kaushlendra T, Sanderson RD, Gaggar A, Richter JR
JCI insight 2022 Aug 8;7(15)

Cheng L, Zhu X, Liu Y, Zhu K, Lin K, Li F
Annals of translational medicine 2021 Sep;9(18):1454

Wu TT, Chen YY, Ho CY, Yeh TC, Sun GC, Tseng CJ, Cheng PW
Antioxidants (Basel, Switzerland) 2021 Jul 6;10(7)

Wu TT, Chen YY, Chang HY, Kung YH, Tseng CJ, Cheng PW
Antioxidants (Basel, Switzerland) 2020 Mar 25;9(4)

Wen Z, Jin K, Shen Y, Yang Z, Li Y, Wu B, Tian L, Shoor S, Roche NE, Goronzy JJ, Weyand CM
Nature immunology 2019 Mar;20(3):313-325

Yin H, Zhao L, Li S, Xu L, Wang Y, Chen H
Frontiers in cellular and infection microbiology 2017;7:423

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Upregulation, Antibody-Peptide Competition and Phosphatase Stripping. Extracts of HepG2 cells untreated (1) or treated with 12 mM Metformin for 24 hours in serum free media (2-6) were resolved by SDS-PAGE on a 10% Tris-glycine gel and transferred to PVDF. The membrane was left untreated (1-5) or treated with lambda phosphatase (6), blocked with a 3% BSA-TBST buffer for one hour at room temperature, and then incubated with the Phospho-AMPK alpha-1,2 (Thr183, Thr172) antibody (Product # 44-1150G) for two hours at room temperature in 3% BSA-TBST buffer, following prior incubation with: no peptide (1,2), the non-phosphopeptide corresponding to the phosphopeptide immunogen (3), a generic phosphothreonine-containing peptide (4), or the phosphopeptide immunogen (5). After washing, the membrane was incubated with goat F(ab’)2 anti-rabbit IgG HRP conjugate (Product # ALI4404) and signals were detected using the Pierce SuperSignal™ method. The data show that only the phosphopeptide corresponding to Phospho-AMPK alpha-1,2 (Thr183, Thr172) completely blocks the signal and that phosphatase stripping eliminates the signal, verifying that the antibody is phosphorylation site-specific. The data also show upregulation of the phospho-signal upon Metformin treatment in this cell system.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Phospho-AMPK alpha 1,2 (Thr183, 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) Rabbit Polyclonal Antibody (Product # 44-1150G) at 1:250 dilution 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 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: Immunohistochemistry analysis of Phospho-AMPK alpha 1,2 (Thr183, Thr172) showing staining in the cytoplasm of paraffin-embedded human brain tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Phospho-AMPK alpha-1,2 (Thr183, Thr172) Rabbit Polyclonal Antibody (Product # 44-1150G) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of AMPK alpha 1 + 2 [pThr172] was done on MDA-MB-231 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with AMPK alpha 1 + 2 [pThr172] Rabbit Polyclonal Antibody (441150G, red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody (A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Supportive validation

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 D3T reduces fructose-induced EMT progression in the lens of rats with fructose-induced type 2 DM. ( A ) Immunoblotting analysis depicting Aldose reductase, p-AMPK T172 and Ac-SOD2 expression in the fructose-induced type 2 DM lens with or without D3T administration. ( B ) Immunoblotting analysis showing Snail and Slug protein expressions in the fruc Table 2 . DM lens with or without D3T administration. ( C ) Protein expressions of E-cadherin and vimentin were also analyzed and quantified. All data represented as mean +- SEM ( n = 6 per group, independent experiments in each figure).