PRKAA1 Antibody, HRP conjugated

What is PRKAA1 and what role does it play in cellular function?

PRKAA1 (protein kinase AMP-activated catalytic subunit alpha 1) is the gene encoding the catalytic α1 subunit of AMP-activated protein kinase (AMPK). This protein belongs to the serine/threonine protein kinase family and serves as a critical cellular energy sensor conserved across all eukaryotic cells. AMPK responds to increases in the cellular AMP/ATP ratio by regulating key metabolic enzymes through phosphorylation, effectively protecting cells from energy-depleting stresses by switching off ATP-consuming biosynthetic pathways .

The kinase activity of AMPK, including its PRKAA1 subunit, is particularly important in:

• Regulating cellular energy homeostasis

• Responding to metabolic stress conditions

• Modulating insulin sensitivity

• Influencing cell proliferation and apoptosis pathways

What are the primary applications for PRKAA1 Antibody, HRP conjugated?

PRKAA1 Antibody with HRP conjugation is validated for multiple research applications:

The HRP (Horseradish Peroxidase) conjugation offers direct enzymatic detection capabilities, eliminating the need for secondary antibody incubation steps and thereby streamlining experimental workflows .

What reactivity does the PRKAA1 Antibody, HRP conjugated typically demonstrate?

Commercial PRKAA1 antibodies with HRP conjugation demonstrate reactivity across multiple species:

• Human: Fully validated across multiple cell lines and tissue types

• Mouse: Confirmed cross-reactivity for comparative studies

• Rat: Validated for neurological and metabolic research applications

This multi-species reactivity makes these antibodies particularly valuable for comparative studies and translational research where findings need to be validated across different model systems.

What are the critical factors in optimizing Western blot protocols with PRKAA1 Antibody, HRP conjugated?

Successful Western blot detection of PRKAA1 using HRP-conjugated antibodies requires attention to several technical parameters:

Sample Preparation:

• Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate) if phosphorylated forms of PRKAA1 are of interest

• Optimal protein loading is typically 2 μg/lane as validated in reference studies with 293 cell lysates

Blocking and Antibody Incubation:

• For total PRKAA1 detection, 5% non-fat milk in TBS-T is generally effective

• For phosphorylated PRKAA1 detection, 5% BSA is preferred to avoid interference from phospho-proteins in milk

• Optimal antibody dilution ranges from 1:100-1000, with higher dilutions reducing background

Detection Strategies:

• TMB substrate offers effective visualization with blue color development measured at 600 nm

• For enhanced sensitivity, the reaction can be stopped with hydrochloric acid for measurement at 450 nm

• For lower abundance targets, enhanced chemiluminescence substrates may offer improved sensitivity

How can researchers differentiate between PRKAA1 (AMPKα1) and PRKAA2 (AMPKα2) isoforms?

Distinguishing between the closely related PRKAA1 and PRKAA2 isoforms requires careful antibody selection and validation:

Antibody Selection Criteria:

• Verify the immunogen used for antibody production, with C-terminal epitopes (residues 474-502) typically offering greater isoform specificity

• Consider the subtle molecular weight differences (PRKAA1: 64 kDa; PRKAA2: 62 kDa) when interpreting bands

Validation Approaches:

• Perform side-by-side testing with recombinant PRKAA1 and PRKAA2 proteins

• Include siRNA/shRNA knockdown controls specific to each isoform

• In tissues expressing both isoforms, utilize higher antibody dilutions to minimize cross-reactivity

For definitive isoform identification in complex samples, researchers may need to supplement antibody-based detection with:

• Isoform-specific qPCR for mRNA expression analysis

• Mass spectrometry-based proteomics for unambiguous protein identification

What considerations should be made when using phospho-specific versus total PRKAA1 antibodies?

When employing phospho-specific PRKAA1 antibodies (such as for S487) compared to total PRKAA1 detection, researchers should consider:

Sample Handling:

• Immediate sample processing with phosphatase inhibitors is essential for phospho-epitope preservation

• Flash-freezing tissues immediately upon collection prevents post-collection dephosphorylation

Experimental Design:

• Paired experiments with both phospho-specific and total PRKAA1 antibodies enable calculation of phosphorylation/total protein ratios

• Include positive controls with known phosphorylation status, such as cells treated with AMPK activators (AICAR, metformin) or inhibitors

Interpretational Aspects:

• Different phosphorylation sites have opposing effects on AMPK activity:

  • S487 phosphorylation is inhibitory to AMPK activity

  • T172 phosphorylation (in the activation loop) is activating

• Changes in phosphorylation should be interpreted in the context of upstream kinases (Akt, PKA) and cellular energy status

How is PRKAA1 implicated in cancer development and progression?

PRKAA1 plays complex roles in cancer biology that researchers continue to investigate:

Expression Patterns:

• PRKAA1 shows variable expression across different cancer types and cell lines

• In gastric cancer, high PRKAA1 expression has been observed in BGC-823 and MKN45 cell lines, with lower expression in SGC-7901 and MGC-803 cells

Functional Impact:

• PRKAA1 downregulation by shRNA or AMPK inhibitor compound C has been shown to significantly inhibit proliferation while promoting cell cycle arrest and apoptosis in gastric cancer cells

• PRKAA1 silencing reduced the expression of proliferation marker PCNA and anti-apoptotic protein Bcl-2

Signaling Pathways:

• PRKAA1 promotes gastric cancer cell proliferation and inhibits apoptosis through activation of JNK1 and Akt signaling pathways

• Inactivation of JNK1 or Akt signaling significantly inhibits PRKAA1 overexpression-induced increases in cell proliferation

These findings suggest PRKAA1 may serve as both a potential biomarker and therapeutic target in certain cancers, highlighting the importance of accurate detection using validated antibodies.

What methodological approaches are recommended for PRKAA1 detection in cancer tissues?

For effective PRKAA1 detection in cancer tissues, researchers should consider these methodological approaches:

Immunohistochemistry (IHC):

• Formalin-fixed paraffin-embedded tissues require optimized antigen retrieval, typically using citrate buffer (pH 6.0)

• HRP-conjugated PRKAA1 antibodies can be applied at 1:100-500 dilution followed by DAB staining for visualization

• This approach has been validated in breast carcinoma tissue sections

Tissue Sample Considerations:

• Include both tumor and adjacent normal tissues for comparative analysis

• Consider tumor heterogeneity by examining multiple regions of larger tumors

• Document clinical characteristics to enable correlation with PRKAA1 expression patterns

Quantification Methods:

• Implement digital image analysis for objective quantification of staining intensity

• Use established scoring systems (e.g., H-score, Allred score) for standardized reporting

• Include pathologist verification of staining patterns and cellular localization

For translational significance, correlate PRKAA1 expression with clinical parameters such as tumor stage, grade, and patient outcomes to establish potential prognostic or predictive value.

What are the comparative advantages of ELISA versus Western blotting for PRKAA1 quantification?

Researchers have multiple options for quantitative analysis of PRKAA1 expression, each with distinct advantages:

ELISA-Based Quantification:

• AMPK alpha 1 ELISA kits offer higher throughput and precision for absolute quantification

• The sandwich ELISA format employs a capture antibody and an HRP-conjugated detection system

• Typical detection range spans 0.1-10 ng/mL with systematic sample preparation requirements

Western Blot Quantification with HRP-Conjugated Antibodies:

• Enables simultaneous assessment of protein size, modifications, and relative expression

• Recommended dilutions of 1:100-1000 provide optimal signal-to-noise ratio

• Requires normalized densitometric analysis against loading controls

Comparative Performance:

For most accurate quantification, researchers should select the method that best aligns with their specific experimental questions and available sample material .

How can researchers establish appropriate controls for PRKAA1 antibody validation?

Rigorous validation of PRKAA1 antibody specificity requires implementation of multiple control strategies:

Positive Controls:

• Cell lines with known high PRKAA1 expression (BGC-823, MKN45)

• Recombinant PRKAA1 protein for standard curve generation

• Tissues with documented PRKAA1 expression (liver, skeletal muscle)

Negative Controls:

• PRKAA1 knockout/knockdown samples generated via CRISPR/Cas9 or RNAi

• Non-transfected control lysates run alongside PRKAA1-transfected samples

• Primary antibody omission controls to assess non-specific binding

Specificity Controls:

• Peptide competition assays using the immunizing peptide

• Comparison with alternative antibody clones targeting different epitopes

• Parallel analysis with antibodies specific for PRKAA2 to ensure isoform specificity

The implementation of these controls should be documented to establish the validity and reliability of experimental findings, particularly for publication-quality research.

What strategies should be employed when troubleshooting weak or non-specific signals?

When encountering challenges with PRKAA1 antibody performance, researchers should implement a systematic troubleshooting approach:

For Weak or Absent Signals:

• Increase antibody concentration within recommended range (1:100-500 for IHC, 1:100-1000 for WB)

• Ensure adequate protein loading (minimum 2 μg/lane for Western blots)

• For FFPE tissues, optimize antigen retrieval methods and duration

• Consider more sensitive detection systems (enhanced chemiluminescence)

• Verify sample preparation protocols maintain protein integrity

For Non-specific Signals:

• Increase blocking concentration (5-10% BSA or milk) and duration (1-2 hours)

• Implement more stringent washing (increase PBST concentration to 0.1-0.3% Tween-20)

• Use higher antibody dilutions to reduce non-specific binding

• Consider pre-adsorbing antibody with non-target tissues/lysates

• Verify whether signals are from related proteins using isoform-specific controls

Technical Considerations:

• Check antibody storage conditions—HRP conjugates are particularly sensitive to repeated freeze-thaw cycles

• Verify substrate viability and development conditions

• Consider lot-to-lot variability in antibody performance

What are the optimal storage and handling conditions for maintaining PRKAA1 Antibody, HRP conjugated activity?

Proper storage and handling are critical for maintaining the activity of HRP-conjugated antibodies:

Storage Recommendations:

• Short-term (up to one month): 2-8°C (refrigerated)

• Long-term: -20°C in aliquots to minimize freeze-thaw cycles

• Expected stability when properly stored: 12 months from shipment date

Working Solution Handling:

• Prepare working dilutions on the day of the experiment

• If diluted antibody must be stored, add protein stabilizers (0.5% BSA)

• Protect from extended light exposure which can affect HRP activity

Transportation Considerations:

• Typically shipped on blue ice and should be stored immediately upon receipt

• Brief exposure to ambient temperatures during handling will not significantly impact performance

Following these evidence-based storage guidelines will help ensure consistent antibody performance across experiments and maximize the usable lifespan of these valuable reagents.

How is PRKAA1 antibody detection contributing to metabolic research and AMPK pathway studies?

PRKAA1 Antibody with HRP conjugation serves as a critical reagent in advancing our understanding of AMPK pathway regulation:

AMPK as Metabolic Sensor:

• Detection of PRKAA1 enables researchers to monitor how this catalytic subunit responds to various metabolic conditions such as glucose deprivation, exercise, and hypoxia

• The antibody facilitates studies on how AMP/ATP ratio changes activate AMPK and trigger downstream responses

Disease Model Applications:

• PRKAA1 detection in tissues from metabolic disorder models (diabetes, obesity) reveals alterations in AMPK signaling

• Comparative studies between normal and pathological states can identify potential therapeutic targets

Therapeutic Investigation:

• The antibody enables assessment of how AMPK-targeting drugs (metformin, AICAR) impact PRKAA1 activation

• Researchers can correlate PRKAA1 modulation with metabolic outcomes in cellular and animal models

These applications highlight the central role of PRKAA1 antibodies in advancing our understanding of fundamental metabolic regulation and disease mechanisms.

What novel approaches are being developed for multiplexed detection of AMPK pathway components?

Emerging technologies are enhancing our ability to study PRKAA1 in the context of the broader AMPK signaling network:

Multiplex Immunoassays:

• Simultaneous detection of total and phospho-PRKAA1 alongside other AMPK subunits (β, γ) and downstream targets

• Bead-based platforms allow quantification of multiple proteins from limited sample volumes

Advanced Imaging Approaches:

• Multiplexed immunofluorescence using spectral unmixing to simultaneously visualize PRKAA1 with other pathway components

• Super-resolution microscopy revealing subcellular localization patterns of PRKAA1 under different metabolic conditions

Single-Cell Analysis:

• Mass cytometry (CyTOF) with metal-tagged antibodies enabling high-parameter analysis of AMPK pathway at single-cell resolution

• Single-cell Western blotting techniques for protein-level heterogeneity assessment in complex tissues

Integration with Other Omics:

• Combined proteomics and phosphoproteomics workflows identifying novel PRKAA1 interactions and substrates

• Spatial transcriptomics correlated with protein expression providing tissue context to AMPK signaling

These advanced approaches are expanding our understanding of AMPK biology beyond what traditional single-antibody techniques can reveal.