Mouse PRKAA1 knockout RAW 264.7 cell lysate (ab280114)
Overview
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Product name
Mouse PRKAA1 knockout RAW 264.7 cell lysate -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
RAW 264.7 -
Organism
Mouse -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab280157 - Human PRKAA1 knockout RAW 264.7 cell lysate 1 x 100µg ab277354 - Mouse wild-type RAW 264.7 cell lysate 1 x 100µg -
Research areas
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Cell type
leukaemic monocyte macrophage -
Disease
Carcinoma -
Gender
Male
Target
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Function
Catalytic subunit of AMP-activated protein kinase (AMPK), an energy sensor protein kinase that plays a key role in regulating cellular energy metabolism. In response to reduction of intracellular ATP levels, AMPK activates energy-producing pathways and inhibits energy-consuming processes: inhibits protein, carbohydrate and lipid biosynthesis, as well as cell growth and proliferation. AMPK acts via direct phosphorylation of metabolic enzymes, and by longer-term effects via phosphorylation of transcription regulators. Also acts as a regulator of cellular polarity by remodeling the actin cytoskeleton; probably by indirectly activating myosin. Regulates lipid synthesis by phosphorylating and inactivating lipid metabolic enzymes such as ACACA, ACACB, GYS1, HMGCR and LIPE; regulates fatty acid and cholesterol synthesis by phosphorylating acetyl-CoA carboxylase (ACACA and ACACB) and hormone-sensitive lipase (LIPE) enzymes, respectively. Regulates insulin-signaling and glycolysis by phosphorylating IRS1, PFKFB2 and PFKFB3. AMPK stimulates glucose uptake in muscle by increasing the translocation of the glucose transporter SLC2A4/GLUT4 to the plasma membrane, possibly by mediating phosphorylation of TBC1D4/AS160. Regulates transcription and chromatin structure by phosphorylating transcription regulators involved in energy metabolism such as CRTC2/TORC2, FOXO3, histone H2B, HDAC5, MEF2C, MLXIPL/ChREBP, EP300, HNF4A, p53/TP53, SREBF1, SREBF2 and PPARGC1A. Acts as a key regulator of glucose homeostasis in liver by phosphorylating CRTC2/TORC2, leading to CRTC2/TORC2 sequestration in the cytoplasm. In response to stress, phosphorylates 'Ser-36' of histone H2B (H2BS36ph), leading to promote transcription. Acts as a key regulator of cell growth and proliferation by phosphorylating TSC2, RPTOR and ATG1: in response to nutrient limitation, negatively regulates the mTORC1 complex by phosphorylating RPTOR component of the mTORC1 complex and by phosphorylating and activating TSC2. In response to nutrient limitation, promotes autophagy by phosphorylating and activating ULK1. AMPK also acts as a regulator of circadian rhythm by mediating phosphorylation of CRY1, leading to destabilize it. May regulate the Wnt signaling pathway by phosphorylating CTNNB1, leading to stabilize it. Also has tau-protein kinase activity: in response to amyloid beta A4 protein (APP) exposure, activated by CAMKK2, leading to phosphorylation of MAPT/TAU; however the relevance of such data remains unclear in vivo. Also phosphorylates CFTR, EEF2K, KLC1, NOS3 and SLC12A1. -
Sequence similarities
Belongs to the protein kinase superfamily. CAMK Ser/Thr protein kinase family. SNF1 subfamily.
Contains 1 protein kinase domain. -
Domain
The AIS (autoinhibitory sequence) region some sequence similarity with the ubiquitin-associated domains and represses kinase activity. -
Post-translational
modificationsUbiquitinated.
Phosphorylated at Thr-183 by STK11/LKB1 in complex with STE20-related adapter-alpha (STRADA) pseudo kinase and CAB39. Also phosphorylated at Thr-183 by CAMKK2; triggered by a rise in intracellular calcium ions, without detectable changes in the AMP/ATP ratio. CAMKK1 can also phosphorylate Thr-183, but at a much lower level. Dephosphorylated by protein phosphatase 2A and 2C (PP2A and PP2C). Phosphorylated by ULK1 and ULK2; leading to negatively regulate AMPK activity and suggesting the existence of a regulatory feedback loop between ULK1, ULK2 and AMPK. -
Cellular localization
Cytoplasm. Nucleus. In response to stress, recruited by p53/TP53 to specific promoters. - Information by UniProt
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Alternative names
- 5''-AMP-activated protein kinase catalytic subunit alpha-1
- AAPK1_HUMAN
- ACACA kinase
see all
Associated products
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab280114 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
| Application | Abreviews | Notes |
|---|---|---|
| WB |
Use at an assay dependent concentration. Predicted molecular weight: 64 kDa.
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| Notes |
|---|
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WB
Use at an assay dependent concentration. Predicted molecular weight: 64 kDa. |
Images
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Western Blot
Lane 1: Wild-type RAW 264.7 cell lysate 20 µg
Lane 2: PRKAA1 knockout RAW 264.7 cell lysate 20 µg
Lane 3: Mouse Liver cell lysate 20 µg
Lane 4: Neuro2A cell lysate 20 µg
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.
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Western BlotLane 1: Wild-type RAW 264.7 cell lysate 20µg.
Lane 2: PRKAA1 knockout RAW 264.7 cell lysate 20 µg.
Lane 3: Mouse Liver cell lysate 20 µg.
Lane 4: Neuro2A cell lysate 20 µg.
False colour image of Western blot: Anti-AMPK alpha 1 antibody [2B7] staining at 1/500 dilution, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution shown in red. In Western blot ab110036 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.
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Sanger sequencingMouse Prkaa1 KO in Raw264.7 Cells with 2 bp deletion in Exon 4
Protocols
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.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab280114 has not yet been referenced specifically in any publications.