PRKAA1 (Ab-174/172) Antibody

What is the PRKAA1 (Ab-174/172) Antibody and what epitope does it recognize?

The PRKAA1/PRKAA2 (Ab-174/172) Antibody is a rabbit polyclonal antibody that specifically recognizes the phosphorylated threonine residues at positions 174 and 172 of AMPKα1 and AMPKα2, respectively. The antibody binds to the peptide sequence around (L-R-T-S-C) derived from human AMPKα1/AMPKα2 . This antibody is useful for detecting endogenous levels of PRKAA1/PRKAA2 when phosphorylated at these specific sites, which is critical for monitoring AMPK activation status in various experimental contexts .

The PRKAA1 (Ab-174/172) Antibody has confirmed reactivity with:

• Human

• Mouse

• Rat

Additional predicted reactivity (though not explicitly tested) may include:

• Pig

• Sheep

• Dog

• Cow

• Horse

• Chicken

Cross-reactivity testing is recommended when using with species not explicitly mentioned in the validation data .

What are the optimal storage conditions for maintaining antibody efficacy?

For optimal preservation of antibody activity:

• Store the antibody at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles, as each cycle can significantly reduce binding activity (up to 50% loss per cycle)

• For frequent use, store small working aliquots at -20°C

• The antibody is typically supplied in phosphate-buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol

• Short-term storage at 4°C is acceptable for approximately one week

When handled properly according to these recommendations, the antibody typically maintains its reactivity for 12 months .

What controls should be included when using PRKAA1 (Ab-174/172) Antibody in Western blotting experiments?

For rigorous Western blotting experiments with the PRKAA1 (Ab-174/172) Antibody, include the following controls:

• Positive control: Lysates from cells known to express phosphorylated AMPK (e.g., cells treated with AMPK activators like AICAR or metformin)

• Negative control:

  • Lysates from cells treated with phosphatase

  • Lysates from PRKAA1/PRKAA2 knockout cells

  • Samples treated with lambda phosphatase to remove phosphorylation

• Loading control: Antibodies against housekeeping proteins (β-actin, GAPDH) to ensure equal loading

• Secondary antibody-only control: To identify any non-specific binding from the secondary antibody

• Blocking peptide control: Pre-incubating the antibody with the immunizing peptide sequence (L-R-T-S-C) to confirm specificity

The expected molecular weight for PRKAA1/PRKAA2 is approximately 63 kDa .

How should samples be prepared for optimal detection with this antibody?

For optimal detection using the PRKAA1 (Ab-174/172) Antibody, prepare samples as follows:

For Western Blotting:

• Extract proteins using standard lysis buffers containing phosphatase inhibitors (critical for preserving phosphorylation status)

• Add protease inhibitors to prevent degradation

• Maintain cold temperatures throughout sample preparation

• Separate proteins using 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

• Block with 5% BSA (preferred over milk for phospho-antibodies)

• Use antibody at 1:500-1:1000 dilution

For Immunohistochemistry:

• Fix tissues in 10% neutral buffered formalin

• Embed in paraffin and section (4-6 μm thickness)

• Perform antigen retrieval (typically citrate buffer pH 6.0)

• Block endogenous peroxidase activity

• Use antibody at 1:50-1:100 dilution

For Immunofluorescence:

• Fix cells using 4% paraformaldehyde

• Permeabilize with 0.1-0.3% Triton X-100

• Block with 1-5% BSA or normal serum

• Apply antibody at 1:100-1:200 dilution

What are common issues when using PRKAA1 (Ab-174/172) Antibody in Western blots and how can they be resolved?

How can immunohistochemistry (IHC) protocols be optimized for PRKAA1 (Ab-174/172) Antibody?

To optimize IHC protocols for PRKAA1 (Ab-174/172) Antibody:

• Antigen retrieval optimization:

  • Test multiple methods: citrate buffer (pH 6.0), EDTA buffer (pH 8.0-9.0), or enzymatic retrieval

  • Try different heating times (10-30 minutes)

  • Compare microwave, pressure cooker, and water bath methods

• Antibody dilution optimization:

  • Test a range around the recommended 1:50-1:100 dilution

  • Prepare a dilution series (e.g., 1:25, 1:50, 1:100, 1:200)

  • Select the dilution that gives specific staining with minimal background

• Signal amplification considerations:

  • For weak signals, consider using polymer-based detection systems

  • Biotin-streptavidin systems may provide greater sensitivity

  • Tyramide signal amplification for very low abundance targets

• Counterstaining optimization:

  • Adjust hematoxylin intensity to maintain visibility of positive staining

  • Consider nuclear stains like DAPI for dual visualization with IF

Always include positive and negative control tissues when optimizing conditions.

What factors might affect phospho-specific detection with this antibody?

Several factors can influence phospho-specific detection when using the PRKAA1 (Ab-174/172) Antibody:

• Sample handling:

  • Rapid tissue/cell preservation is critical as phosphorylation states change quickly

  • Flash freezing of tissues minimizes phosphatase activity

  • Avoid repeated freeze-thaw cycles

• Phosphatase inhibitors:

  • Always include multiple phosphatase inhibitors in lysis buffers (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate, and phosphatase inhibitor cocktails)

  • Prepare inhibitors fresh for each experiment

• Blocking reagents:

  • BSA is preferred over milk for phospho-antibodies (milk contains phosphoproteins)

  • Use 3-5% BSA in TBS-T for blocking and antibody dilution

• Cell culture conditions:

  • Cellular stress during harvesting can alter phosphorylation status

  • Serum starvation can affect baseline AMPK phosphorylation

  • Confluence level impacts AMPK activation

• Drug treatments:

  • Consider timing carefully when using AMPK activators or inhibitors

  • Document exposure duration precisely for reproducibility

How can the PRKAA1 (Ab-174/172) Antibody be used to investigate the role of AMPK in cancer progression?

Recent research has highlighted PRKAA1's significant role in cancer biology, making this antibody valuable for investigating:

• Prognostic biomarker analysis:

  • Use IHC with the PRKAA1 (Ab-174/172) Antibody on tissue microarrays to correlate phospho-AMPK expression with patient outcomes

  • Combine with other markers to develop multi-parameter prognostic panels

  • Quantify staining intensity and distribution using digital pathology platforms

• Tumor metabolism studies:

  • Compare AMPK phosphorylation states between tumor and adjacent normal tissues

  • Correlate with metabolic enzyme expression patterns

  • Track changes during metastatic progression

• Immunotherapy response prediction:

  • Recent pan-cancer analysis has shown PRKAA1 expression correlates with immunotherapy response

  • This antibody can help identify phospho-AMPK status in pre-treatment biopsies

  • May help stratify patients for clinical trials

• PI3K/AKT pathway interaction:

  • Research indicates PRKAA1 influences cancer progression through the PI3K/AKT pathway

  • Use this antibody alongside phospho-AKT antibodies to map pathway interactions

  • Test sensitivity to AKT inhibitors (MK2206, GSK2110183) in relation to PRKAA1 phosphorylation status

For these advanced applications, quantitative analysis methods like multiplex immunofluorescence may provide deeper insights than traditional single-marker approaches.

What methodologies enable temporal monitoring of AMPK activation in live cell systems?

While the PRKAA1 (Ab-174/172) Antibody is primarily designed for fixed samples, researchers can implement complementary approaches for temporal monitoring:

• Sequential sampling with phospho-specific detection:

  • Harvest cells at multiple time points after treatment

  • Process immediately for Western blotting with PRKAA1 (Ab-174/172) Antibody

  • Plot activation curves based on quantified band intensity

  • Normalize to total AMPK levels using a non-phospho-specific antibody

• Integration with biosensor technologies:

  • Combine fixed-time-point antibody analysis with live-cell FRET-based AMPK biosensors

  • Correlate biosensor signals with antibody-detected phosphorylation levels

  • Use phospho-specific antibody validation at key timepoints to calibrate biosensor readings

• Immunofluorescence time-course microscopy:

  • Fix parallel cell cultures at defined intervals

  • Process with PRKAA1 (Ab-174/172) Antibody (1:100-1:200 dilution)

  • Quantify fluorescence intensity changes across the time series

  • Co-stain with subcellular markers to track localization changes

• Flow cytometry time-course analysis:

  • Fix and permeabilize cells at various timepoints

  • Stain with PRKAA1 (Ab-174/172) Antibody

  • Quantify population-level changes in phosphorylation status

  • Identify responding subcellular populations

What is the physiological significance of PRKAA1/PRKAA2 phosphorylation at Thr174/Thr172?

The phosphorylation of PRKAA1/PRKAA2 at Thr174/Thr172 has critical physiological implications:

• Activation mechanism:

  • Phosphorylation at these threonine residues is essential for AMPK catalytic activity

  • This modification occurs in response to increased cellular AMP:ATP ratio

  • Upstream kinases like LKB1 and CaMKKβ catalyze this phosphorylation

• Metabolic regulation:

  • Activated AMPK regulates fatty acid synthesis by phosphorylating acetyl-CoA carboxylase

  • It inhibits cholesterol synthesis via phosphorylation of hydroxymethylglutaryl-CoA reductase

  • It phosphorylates and inactivates hormone-sensitive lipase

  • Acts as a metabolic stress sensor, shutting down biosynthetic pathways when cellular ATP levels are depleted

• Energy homeostasis:

  • AMPK activation increases glucose uptake and fatty acid oxidation

  • Promotes mitochondrial biogenesis to enhance ATP production

  • Inhibits protein synthesis to conserve energy during stress conditions

• Cellular adaptation:

  • Phosphorylated AMPK responds to fuel limitation and hypoxia

  • Triggers autophagy to recycle cellular components during energy crisis

  • Protects cells from apoptosis during transient metabolic stress

Monitoring the Thr174/Thr172 phosphorylation status using this antibody provides direct insight into cellular energy status and metabolic regulation.

How does PRKAA1/PRKAA2 activation interface with other signaling pathways in cellular stress responses?

PRKAA1/PRKAA2 activation intersects with multiple signaling networks in coordinating cellular stress responses:

• mTOR pathway crosstalk:

  • Phosphorylated AMPK inhibits mTORC1 through direct phosphorylation of TSC2 and Raptor

  • This suppresses protein synthesis and ribosome biogenesis during energy stress

  • Use the PRKAA1 (Ab-174/172) Antibody alongside phospho-mTOR antibodies to map this interaction

• PI3K/AKT pathway interactions:

  • PRKAA1 has been shown to regulate cancer progression through the PI3K/AKT pathway

  • Research indicates cells with high PRKAA1 phosphorylation show differential sensitivity to AKT inhibitors

  • Combined analysis of phospho-AMPK and phospho-AKT provides insights into this crosstalk

• p53 signaling:

  • AMPK phosphorylates p53, promoting cell cycle arrest during metabolic stress

  • This creates a metabolic checkpoint that prevents cell division during energy limitation

  • Co-immunoprecipitation with PRKAA1 (Ab-174/172) Antibody can help identify binding partners

• Insulin signaling pathway:

  • AMPK activation can both enhance and antagonize insulin signaling

  • During energy stress, AMPK promotes GLUT4 translocation, enhancing glucose uptake

  • AMPK can also induce insulin resistance through specific phosphorylation events

  • Multiplex analysis with insulin pathway components helps elucidate these complex interactions

• Inflammatory pathways:

  • AMPK activation generally suppresses NF-κB-mediated inflammatory responses

  • This creates important connections between metabolic status and immune function

  • Research exploring these connections may explain PRKAA1's role in immunotherapy response

What are the emerging roles of PRKAA1 in cancer and immunotherapy response?

Recent research has uncovered important roles for PRKAA1 in cancer biology and immunotherapy:

• Prognostic significance:

  • Pan-cancer analysis has identified PRKAA1 as a potential prognostic marker

  • The PRKAA1 (Ab-174/172) Antibody can be used to evaluate phospho-AMPK status in tumor samples

  • Expression patterns correlate with patient outcomes in various cancer types

• Pancreatic cancer progression:

  • Downregulation of PRKAA1 has been shown to reduce proliferation, migration, and invasion of pancreatic cancer cells

  • Researchers found PRKAA1 regulates pancreatic cancer progression potentially through the PI3K/AKT pathway

  • This suggests PRKAA1 as a potential therapeutic target

• Immunotherapy response prediction:

  • PRKAA1 expression correlates with various immune profiles

  • This relationship suggests PRKAA1 could potentially be used to predict or enhance immunotherapy responses

  • The mechanism may involve AMPK's role in regulating immune cell metabolism and function

• Differential drug sensitivity:

  • Experimental evidence shows cells with PRKAA1 overexpression exhibited reduced sensitivity to AKT inhibitors (MK2206, GSK2110183)

  • This suggests PRKAA1 status could help guide therapeutic decision-making

  • Monitoring phospho-AMPK with this antibody could help identify patients likely to respond to specific treatments

These findings highlight the potential of PRKAA1 (Ab-174/172) Antibody as both a research tool and a potential companion diagnostic for personalized cancer therapy strategies.

What emerging technologies might enhance the utility of phospho-specific AMPK antibodies in metabolic research?

Several cutting-edge technologies could extend the applications of the PRKAA1 (Ab-174/172) Antibody:

• Single-cell proteomics:

  • Integration with microfluidic antibody-based platforms for single-cell resolution of phospho-AMPK

  • Correlation with single-cell transcriptomics to link AMPK activation to gene expression changes

  • Spatial proteomics to map subcellular phospho-AMPK localization in intact tissue samples

• Proximity labeling technologies:

  • BioID or APEX2 fusion proteins combined with phospho-specific antibody validation

  • Mapping the changing interactome of AMPK based on phosphorylation status

  • Identifying novel phosphorylation-dependent protein interactions

• Advanced imaging techniques:

  • Super-resolution microscopy with PRKAA1 (Ab-174/172) Antibody for nanoscale localization

  • Expansion microscopy to physically enlarge specimens for enhanced phospho-AMPK visualization

  • Correlative light and electron microscopy to link phospho-AMPK to ultrastructural features

• In vivo monitoring systems:

  • Intravital microscopy combined with ex vivo antibody validation

  • Non-invasive imaging of AMPK activity correlated with antibody-based tissue analysis

  • Development of antibody-based PET imaging tracers for clinical translation