重组Anti-AMPK alpha 1抗体[Y365] (ab32047)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y365] to AMPK alpha 1
- Suitable for: ICC/IF, Flow Cyt (Intra), WB, IHC-P, IP
- Knockout validated
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
概述
-
产品名称
Anti-AMPK alpha 1抗体[Y365]
参阅全部 AMPK alpha 1 一抗 -
描述
兔单克隆抗体[Y365] to AMPK alpha 1 -
宿主
Rabbit -
特异性
This antibody is specific for human AMPK alpha 1. This antibody shows low affinity on mouse and rat samples. -
经测试应用
适用于: ICC/IF, Flow Cyt (Intra), WB, IHC-P, IPmore details -
种属反应性
与反应: Mouse, Rat, Human
预测可用于: African green monkey -
免疫原
Synthetic peptide within Human AMPK alpha 1 aa 500 to the C-terminus (C terminal). The exact sequence is proprietary.
Database link: Q13131 -
阳性对照
- WB: HeLa, HepG2, C6, NIH/3T3 and MCF-7 cell lysate. Mouse liver, brain, retina, and skeletal muscle tissue lysates, RAW 264.7 cell lysate, Neuro2A cell lysate. IHC-P: Human cervical carcinoma and lung carcinoma tissues. ICC/IF: MCF-7 cells. Flow Cyt (intra): HeLa cells. IP: HeLa whole cell lysate (ab150035).
-
常规说明
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
性能
-
形式
Liquid -
存放说明
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
存储溶液
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
-
纯度
Protein A purified -
克隆
单克隆 -
克隆编号
Y365 -
同种型
IgG -
研究领域
相关产品
-
Alternative Versions
-
Compatible Secondaries
-
Conjugation kits
-
Isotype control
-
Positive Controls
-
Recombinant Protein
-
Related Products
应用
The Abpromise guarantee
Abpromise™承诺保证使用ab32047于以下的经测试应用
“应用说明”部分 下显示的仅为推荐的起始稀释度;实际最佳的稀释度/浓度应由使用者检定。
应用 | Ab评论 | 说明 |
---|---|---|
ICC/IF |
1/250.
|
|
Flow Cyt (Intra) |
1/100 - 1/150.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
|
WB | (5) |
1/1000 - 1/5000. Predicted molecular weight: 63 kDa.
This antibody shows low affinity on mouse and rat samples. |
IHC-P |
1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
See IHC antigen retrieval protocols. |
|
IP |
1/40 - 1/50.
|
说明 |
---|
ICC/IF
1/250. |
Flow Cyt (Intra)
1/100 - 1/150. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
WB
1/1000 - 1/5000. Predicted molecular weight: 63 kDa. This antibody shows low affinity on mouse and rat samples. |
IHC-P
1/100 - 1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. See IHC antigen retrieval protocols. |
IP
1/40 - 1/50. |
靶标
-
功能
Responsible for the regulation of fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. It also regulates cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. Appears to act as a metabolic stress-sensing protein kinase switching off biosynthetic pathways when cellular ATP levels are depleted and when 5'-AMP rises in response to fuel limitation and/or hypoxia. This is a catalytic subunit. -
序列相似性
Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. SNF1 subfamily.
Contains 1 protein kinase domain. - Information by UniProt
-
数据库链接
- Entrez Gene: 5562 Human
- Entrez Gene: 105787 Mouse
- Entrez Gene: 65248 Rat
- Omim: 602739 Human
- SwissProt: Q13131 Human
- SwissProt: Q5EG47 Mouse
- SwissProt: P54645 Rat
- Unigene: 43322 Human
see all -
别名
- 5 AMP activated protein kinase alpha 1catalytic subunit antibody
- 5 AMP activated protein kinase catalytic alpha 1 chain antibody
- 5' AMP activated protein kinase catalytic subunit alpha 1 antibody
see all
图片
-
All lanes : Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/1000 dilution
Lane 1 : Wild-type RAW 264.7 cell lysate
Lane 2 : PRKAA1 knockout RAW 264.7 cell lysate
Lane 3 : Mouse Liver cell lysate
Lane 4 : Neuro2A cell lysate
Lysates/proteins at 20 µg per lane.
Performed under reducing conditions.
Predicted band size: 63 kDa
Observed band size: 64 kDa why is the actual band size different from the predicted?False colour image of Western blot: Anti-AMPK alpha 1 antibody [Y365] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32047 was shown to bind specifically to AMPK alpha 1. A band was observed at 64 kDa in wild-type RAW 264.7 cell lysates with no signal observed at this size in PRKAA1 knockout cell line ab280055 (knockout cell lysate ab280114). To generate this image, wild-type and PRKAA1 knockout RAW 264.7 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged.Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.
-
Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: AMPK alpha knockout HAP1 cell lysate (20 µg)
Lane 3: MCF7 cell lysate (20 µg)
Lane 4: HeLa cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32047 observed at 63 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32047 was shown to specifically react with AMPK alpha in wild-type HAP1 cells. No band was observed when AMPK alpha knockout samples were examined. Wild-type and AMPK alpha knockout samples were subjected to SDS-PAGE. ab32047 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1hr at room temperature before imaging. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AMPK alpha 1 antibody [Y365] (ab32047)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue labelling AMPK alpha 1 with purified ab32047 at 1/100. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9 before commencing with IHC staining protocol. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.
-
Immunocytochemistry/Immunofluorescence analysis of MCF7 cells labelling AMPK alpha 1 with purified ab32047 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500) were also used.
Control 1: primary antibody (1/250) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/500).
Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500).
-
Intracellular Flow Cytometry analysis of HeLa cells labelling AMPK alpha 1 with purified ab32047 at 1/150 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.
-
ab32047 (purified) at 1/40 immunoprecipitating AMPK alpha 1 in HeLa whole cell lysate.
Lane 1 (input): HeLa whole cell lysate (10µg)
Lane 2 (+): ab32047 + HeLa whole cell lysate (10µg).
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32047 in HeLa whole cell lysate.
For western blotting, ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection (1/1500).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
-
All lanes : Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/500 dilution
Lane 1 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lane 3 : Mouse liver tissue lysate
Lane 4 : Mouse brain tissue lysate
Lane 5 : Mouse kidney tissue lysate
Lane 6 : Mouse retina tissue lysate
Lane 7 : Mouse skeletal muscle tissue lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 63 kDa
Observed band size: 63 kDa
Additional bands at: 40 kDa (possible non-specific binding)
Exposure time: 3 minutesBlocking/Diluting buffer and concentration: 5% NFDM/TBST.
This antibody shows low affinity on mouse samples.
-
All lanes : Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/20000 dilution
Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate
Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 4 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 5 : C6 (Rat glial tumor glial cell) whole cell lysate
Lane 6 : Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysate
Lane 7 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 63 kDa
Observed band size: 63 kDa
Exposure time: 180 secondsBlocking and dilution buffer and concentration: 5% NFDM/TBST.
This antibody shows low affinity on mouse and rat samples.
-
All lanes : Anti-AMPK alpha 1 antibody [Y365] (ab32047) at 1/2000 dilution (purified)
Lane 1 : C6 cell lysate
Lane 2 : NIH/3T3 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Peroxidase-conjugated goat anti-rabbit IgG, (H+L) at 1/1000 dilution
Predicted band size: 63 kDa
Observed band size: 63 kDaBlocking and dilution buffer: 5% NFDM/TBST.
This antibody shows low affinity on mouse and rat samples. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-AMPK alpha 1 antibody [Y365] (ab32047)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analaysis of human cervical carcinoma tissue labelling AMPK alpha 1 with unpurified ab32047 at a dilution of 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
-
Overlay histogram showing HeLa cells stained with unpurified ab32047 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab32047, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.
实验方案
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
数据表及文件
-
SDS download
-
Datasheet download
文献 (165)
ab32047 被引用在 165 文献中.
- Ji P et al. Genetically engineered probiotics as catalytic glucose depriver for tumor starvation therapy. Mater Today Bio 18:100515 (2023). PubMed: 36582449
- Yang J et al. Pitavastatin activates mitophagy to protect EPC proliferation through a calcium-dependent CAMK1-PINK1 pathway in atherosclerotic mice. Commun Biol 5:124 (2022). PubMed: 35145192
- Chen M et al. Metformin protects lens epithelial cells against senescence in a naturally aged mouse model. Cell Death Discov 8:8 (2022). PubMed: 35013152
- Zhang X et al. Exogenous Linoleic Acid Intervention Alters Hepatic Glucose Metabolism in an Avian Embryo Model. Front Physiol 13:844148 (2022). PubMed: 35264980
- Niemann B et al. AMPK Activation Is Indispensable for the Protective Effects of Caloric Restriction on Left Ventricular Function in Postinfarct Myocardium. Biology (Basel) 11:N/A (2022). PubMed: 35336822