PRKAB1 Monoclonal Antibody

What is PRKAB1 and why is it an important research target?

PRKAB1 (Protein Kinase, AMP-Activated, beta 1 Non-Catalytic Subunit) encodes the β1 regulatory subunit of AMP-activated protein kinase (AMPK). AMPK functions as a critical energy-sensing enzyme that monitors cellular energy status through a heterotrimeric complex consisting of a catalytic α subunit and regulatory β and γ subunits. The β1 subunit serves as a positive regulator of AMPK activity and may function as an adaptor molecule mediating the association of the AMPK complex. PRKAB1 is significant in research because of its role in cellular energy homeostasis, particularly in response to metabolic stresses when AMPK is activated to phosphorylate and inactivate acetyl-CoA carboxylase and β-hydroxy β-methylglutaryl-CoA reductase, which are key enzymes involved in regulating fatty acid and cholesterol biosynthesis .

What applications are PRKAB1 monoclonal antibodies validated for?

PRKAB1 monoclonal antibodies are validated for multiple research applications including:

The specific monoclonal antibody against AMPK Beta 1 (1A7) reacts with human, monkey, mouse, and rat PRKAB1, making it versatile for comparative studies across these species .

How does one properly store and handle PRKAB1 monoclonal antibodies?

For optimal performance and longevity of PRKAB1 monoclonal antibodies, adhere to these storage and handling guidelines:

For long-term storage, maintain antibodies at -20°C for up to one year in their original formulation. If frequent usage is anticipated, store at 4°C for up to one month. The antibody is typically supplied as 1mg/ml in PBS with 0.02% sodium azide and 50% glycerol at pH 7.2. Repeated freeze-thaw cycles should be strictly avoided as they can cause protein denaturation and aggregation, significantly reducing antibody performance. When handling, always use clean pipette tips and sterile technique to prevent contamination .

How should researchers design experiments to validate PRKAB1 antibody specificity?

Designing robust validation experiments for PRKAB1 antibody specificity requires a multi-faceted approach:

• Positive and negative controls: Include cell lysates known to express PRKAB1 (e.g., JURKAT, PANC, K562 cell lines) as positive controls. For negative controls, use either PRKAB1 knockdown/knockout samples or cells known not to express the protein.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide used to generate it, which should abolish specific binding. This is particularly valuable for polyclonal antibodies.

• Cross-reactivity testing: Evaluate potential cross-reactivity with related proteins, particularly PRKAB2 (the β2 subunit), as confirmed by the manufacturer stating "no cross-reactivity with other proteins" .

• Molecular weight verification: PRKAB1 has a calculated molecular weight of approximately 30.4 kDa, though it typically appears around 38 kDa on SDS-PAGE due to post-translational modifications. Confirm that the detected band aligns with the expected molecular weight .

• Multiple detection methods: Verify antibody specificity using different techniques such as Western blot, immunohistochemistry, and immunofluorescence to ensure consistent results across platforms.

What controls should be included when using phospho-specific PRKAB1 antibodies?

When working with phospho-specific antibodies such as anti-PRKAB1 (pSer108), implementing appropriate controls is critical for result interpretation:

• Treatment controls: Include samples with treatments known to induce or inhibit phosphorylation:

  • AMPK activators (e.g., AICAR, metformin) to increase phosphorylation

  • AMPK inhibitors (e.g., Compound C) to decrease phosphorylation

  • Lambda phosphatase-treated samples to remove all phosphorylation

• Paired antibodies: Run parallel blots with phospho-specific and total PRKAB1 antibodies to calculate the phosphorylation ratio and normalize for protein expression variations.

• Specificity verification: Confirm that the antibody recognizes only the phosphorylated form (pSer108) of PRKAB1 and not the unphosphorylated form, using synthetic phosphorylated peptides as competitors .

• Cross-specificity testing: Ensure the antibody does not cross-react with other phosphorylated proteins, particularly those with similar phosphorylation motifs (such as TRSHN in the case of pSer108) .

• Temporal controls: Include time-course samples to demonstrate dynamic phosphorylation changes following relevant stimuli.

How can researchers optimize immunofluorescence protocols using PRKAB1 antibodies?

For optimal immunofluorescence results with PRKAB1 antibodies, consider the following methodological refinements:

• Fixation optimization: Test both formaldehyde (4%) and methanol fixation methods, as the preservation of PRKAB1 epitopes may vary depending on the antibody. For the polyclonal PRKAB1 antibody, enzymatic antigen retrieval has been successfully employed .

• Blocking conditions: Use 10% serum (from the species in which the secondary antibody was raised) in PBS with 0.1-0.3% Triton X-100 for permeabilization. This combination reduces background while maintaining specific signal .

• Antibody concentration titration: Begin with the manufacturer's recommended dilution (typically 2μg/mL for PRKAB1 antibodies) and test a range above and below to determine optimal signal-to-noise ratio.

• Incubation conditions: Overnight incubation at 4°C often produces better results than shorter incubations at room temperature, enhancing specific binding while minimizing background.

• Signal amplification: Consider using fluorophore-conjugated secondary antibodies with matching filters (e.g., DyLight®488 Conjugated Goat Anti-Rabbit IgG) at 1:100 dilution with 30-minute incubation at 37°C for optimal visualization .

• Counterstaining: Use DAPI for nuclear visualization to provide cellular context for PRKAB1 localization. This helps distinguish between cytoplasmic, nuclear, or membrane-associated staining patterns.

What are the common causes of non-specific binding when using PRKAB1 antibodies in Western blotting?

Non-specific binding in Western blots using PRKAB1 antibodies can arise from several factors:

• Insufficient blocking: Inadequate blocking may result in antibody binding to non-specific sites. Optimize blocking by using 5% non-fat milk in TBS for at least 1.5 hours at room temperature, as demonstrated in validated protocols .

• Antibody concentration: Excessive antibody concentration can increase background. Start with the recommended dilution (1:1000 for WB) and adjust as needed based on signal-to-noise ratio .

• Cross-reactivity with related proteins: While manufacturers report no cross-reactivity with other proteins, structural similarities between AMPK subunits may cause non-specific binding. Validate using knockout/knockdown controls.

• Sample preparation issues: Incomplete denaturation or improper sample preparation can expose epitopes that cause non-specific binding. Ensure thorough denaturation by heating samples at 95°C for 5 minutes in reducing sample buffer.

• Secondary antibody issues: Non-specific binding may come from the secondary antibody. Test by running a control blot with secondary antibody only.

• Poor membrane washing: Insufficient washing between antibody incubations can leave residual unbound antibodies. Implement three 5-minute washes with TBS-0.1% Tween between each antibody incubation step .

How can researchers troubleshoot weak or absent signals in immunohistochemistry using PRKAB1 antibodies?

When encountering weak or absent signals in IHC with PRKAB1 antibodies, implement the following troubleshooting steps:

• Antigen retrieval optimization: Different antibodies may require specific antigen retrieval methods. For PRKAB1, enzyme-based antigen retrieval has proven effective. Test both heat-induced epitope retrieval (HIER) using citrate or EDTA buffers and enzymatic retrieval to determine optimal conditions .

• Antibody concentration adjustment: Increase antibody concentration incrementally if signal is weak. For polyclonal antibodies, concentrations up to 5μg/mL may be necessary in difficult tissues.

• Incubation time extension: Extending primary antibody incubation from overnight to 48 hours at 4°C can enhance sensitivity for weakly expressed proteins.

• Detection system enhancement: Switch to more sensitive detection systems like polymer-based HRP systems or tyramide signal amplification if standard methods yield weak signals.

• Tissue fixation evaluation: Overfixation can mask epitopes. If possible, test tissues with different fixation durations or consider using frozen sections if formalin-fixed samples consistently yield poor results.

• Endogenous peroxidase blocking: Ensure complete blocking of endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 15 minutes before antibody application.

What strategies can be employed when facing inconsistent results across biological replicates?

Inconsistency across biological replicates when using PRKAB1 antibodies may stem from several sources that can be addressed methodically:

• Standardization of sample preparation: Ensure all samples are processed identically, including lysis buffer composition, protein concentration determination method, and storage conditions. For western blotting, loading 40μg of sample under reducing conditions has been validated for consistent PRKAB1 detection .

• Antibody lot variation: Different antibody lots may have varying affinities. When possible, use the same lot for an entire study, or validate new lots against previous ones before continuing experiments.

• Biological variation in PRKAB1 expression/phosphorylation: AMPK activity is highly responsive to cellular energy status and stress conditions. Standardize cell culture conditions, including confluence, passage number, and serum batches to minimize these variations.

• Protocol consistency: Document and strictly follow standardized protocols for all replicates, including antibody dilutions, incubation times, and washing steps. For instance, maintain consistent electrophoresis conditions (70V for stacking gel, 90V for resolving gel, for 2-3 hours) .

• Internal loading controls: Always include appropriate loading controls (β-actin, GAPDH) for Western blots and reference genes for qPCR to normalize for loading variations.

• Technical normalization: Consider using pooled standards across blots/experiments that can be used to normalize between experimental runs, especially for quantitative comparisons.

How can PRKAB1 phosphorylation at Ser108 be used to assess AMPK activation status in diverse cellular contexts?

PRKAB1 phosphorylation at Ser108 serves as a reliable indicator of AMPK activation status and can be leveraged in various research contexts:

Ser108 phosphorylation is particularly valuable because it precedes and enhances subsequent phosphorylation of Thr172 on the α subunit, which is the primary marker of AMPK activation. By using phospho-specific antibodies targeting pSer108, researchers can detect early activation events in the AMPK pathway .

For comprehensive AMPK activation assessment, implement a multi-marker approach:

• Measure PRKAB1 pSer108 using specific antibodies

• Detect PRKAA1/2 pThr172 as the canonical activation marker

• Assess downstream substrate phosphorylation (e.g., ACC pSer79)

This approach provides temporal insights into activation dynamics, as Ser108 phosphorylation may precede full AMPK activation. The sequence context of Ser108 (TRSHN) is critical for antibody recognition and should be considered when interpreting results across species .

When comparing AMPK activation across different tissues or cell types, be aware that PRKAB1 expression levels vary significantly. Normalization to total PRKAB1 is essential for accurate interpretation of phosphorylation data.

What considerations should be made when designing co-immunoprecipitation experiments to study PRKAB1 interactions?

Co-immunoprecipitation (Co-IP) experiments with PRKAB1 require careful design to preserve physiologically relevant protein interactions:

• Antibody selection: Choose antibodies that recognize native conformations and do not interfere with protein-protein interaction sites. The monoclonal PRKAB1 antibody (1A7) has been validated for immunoprecipitation at dilutions of 1:50-200 .

• Lysis buffer optimization: Use mild, non-denaturing lysis buffers to preserve protein-protein interactions. For AMPK complexes, consider:

  • 50 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 1% NP-40 or 0.5% Triton X-100

  • 1 mM EDTA

  • Protease and phosphatase inhibitors

• Cross-linking consideration: For transient or weak interactions, implement reversible cross-linking with DSP (dithiobis[succinimidyl propionate]) before cell lysis.

• Pre-clearing strategy: Pre-clear lysates with protein A/G beads to reduce non-specific binding, particularly important when using the mouse monoclonal PRKAB1 antibody to prevent direct binding to the beads.

• Negative controls: Include isotype-matched control IgG immunoprecipitations and, when possible, samples from PRKAB1 knockout/knockdown cells.

• Detection strategy: For interacting proteins, consider specific antibodies against known or suspected AMPK complex components (α1, α2, β2, γ1-3) and potential regulatory partners.

• Validation by reciprocal Co-IP: Confirm interactions by performing reverse Co-IP with antibodies against the interacting protein to pull down PRKAB1.

How does PRKAB1 myristoylation affect antibody recognition and experimental design?

PRKAB1 undergoes N-terminal myristoylation, which has significant implications for antibody recognition and experimental approaches:

The myristoylation of PRKAB1 occurs at the N-terminus and affects subcellular localization and AMPK complex formation. This post-translational modification can potentially mask epitopes in this region, particularly affecting antibodies targeting N-terminal sequences .

When selecting antibodies, consider the following:

• Antibodies targeting the N-terminal region (AA 4-34) may have differential recognition of myristoylated versus non-myristoylated forms

• Antibodies recognizing central or C-terminal regions (e.g., AA 201-270) are less likely to be affected by myristoylation status

For experiments specifically investigating myristoylation:

• Use site-directed mutagenesis of the myristoylation site (G2A mutation) as a control

• Compare antibody recognition between wild-type and G2A mutant proteins

• Consider using N-myristoyltransferase inhibitors to modulate myristoylation status

When studying subcellular localization, be aware that myristoylation influences membrane association. Immunofluorescence protocols may need optimization to preserve membrane structures, such as using gentle permeabilization with digitonin rather than stronger detergents like Triton X-100.

How should researchers interpret discrepancies between observed and calculated molecular weights of PRKAB1?

Discrepancies between the calculated molecular weight of PRKAB1 (30.4 kDa) and its observed migration on SDS-PAGE (approximately 38 kDa) are common and scientifically meaningful:

This difference is primarily attributable to post-translational modifications (PTMs) and structural features that affect protein migration. For PRKAB1, these include:

• Phosphorylation: PRKAB1 undergoes phosphorylation at multiple sites including Ser108 and Ser182, which can add approximately 80 Da per phosphate group but may cause disproportionately larger shifts in apparent molecular weight due to changes in protein charge and SDS binding .

• Myristoylation: N-terminal myristoylation adds a 14-carbon fatty acid chain (~210 Da) but may cause a larger shift due to altered SDS binding to the hydrophobic moiety.

• Glycosylation: While not extensively documented for PRKAB1, potential glycosylation would significantly increase observed molecular weight.

• Protein conformation: Incomplete denaturation can result in compact structures that migrate faster than fully denatured proteins.

When encountering unexpected molecular weights:

• Verify antibody specificity using knockout/knockdown controls

• Consider using phosphatase treatment to determine if phosphorylation contributes to the shift

• Compare migration patterns across different cell types or tissues

• Use gradient gels (5-20% SDS-PAGE) for improved resolution of proteins in this size range

What factors influence the interpretation of PRKAB1 phosphorylation data in metabolic stress studies?

When analyzing PRKAB1 phosphorylation in metabolic stress studies, consider these critical factors for accurate interpretation:

• Temporal dynamics: PRKAB1 phosphorylation at Ser108 exhibits distinct temporal patterns following metabolic stress. Initial phosphorylation can occur rapidly (within 5-15 minutes) but may be transient or sustained depending on the nature and duration of the stress. Time-course experiments are essential for comprehensive understanding.

• Energy status context: AMPK activation, including PRKAB1 phosphorylation, is highly sensitive to cellular AMP:ATP and ADP:ATP ratios. Parallel measurements of these nucleotides or proxies of energy status provide crucial context for phosphorylation data.

• Upstream kinase activity: Ser108 phosphorylation can occur via AMPK autophosphorylation or through upstream kinases. This distinction is important when interpreting data from experiments using AMPK inhibitors or activators.

• Isoform compensation: In studies involving PRKAB1 knockdown or knockout, potential compensatory upregulation of PRKAB2 (the β2 isoform) may occur. This compensation can mask phenotypes and should be assessed when interpreting results.

• Tissue/cell-specific regulation: PRKAB1 expression and phosphorylation patterns vary significantly between tissues. For instance, skeletal muscle exhibits different AMPK complex composition and regulation compared to liver or adipose tissue.

• Normalization approach: When quantifying phosphorylation, normalization to total PRKAB1 rather than housekeeping proteins provides more accurate assessment of the phosphorylation stoichiometry independent of expression changes.

How can researchers integrate PRKAB1 phosphorylation data with broader AMPK signaling networks?

Integrating PRKAB1 phosphorylation data into the broader context of AMPK signaling requires a systems biology approach:

• Multi-site phosphorylation analysis: AMPK activation involves phosphorylation of multiple sites across different subunits. Create a comprehensive phosphorylation profile by analyzing:

  • PRKAB1 pSer108 (regulatory, enhances kinase activity)

  • PRKAA1/2 pThr172 (primary activation marker in α subunit)

  • PRKAB1 pSer182 (less studied, potentially regulatory)

  • Downstream substrate phosphorylation (ACC, ULK1, TSC2)

• Pathway cross-talk evaluation: AMPK signaling intersects with multiple pathways including mTOR, insulin signaling, and autophagy. Analyze key nodes in these pathways (e.g., S6K, Akt, LC3) alongside PRKAB1 phosphorylation.

• Temporal resolution: Implement time-resolved experiments to distinguish between primary AMPK-mediated effects and secondary adaptive responses, particularly when studying metabolic reprogramming.

• Computational modeling: For complex datasets, employ mathematical modeling approaches:

  • Ordinary differential equation (ODE) models for temporal dynamics

  • Bayesian networks for inferring causal relationships

  • Principal component analysis for identifying major sources of variation

• Multi-omics integration: Combine phosphoproteomics data with:

  • Transcriptomics to identify gene expression changes

  • Metabolomics to correlate with metabolic adaptations

  • Interactomics to map dynamic protein-protein interactions

• Functional validation: Test model predictions through targeted interventions:

  • Phosphomimetic and phosphodeficient PRKAB1 mutants

  • Isoform-specific knockdown/knockout

  • Pharmacological modulation with varying temporal patterns

This integrated approach transforms isolated phosphorylation data into mechanistic insights regarding AMPK's role in cellular energy homeostasis and stress responses.

What are the methodological considerations for studying PRKAB1 in primary tissues versus cell lines?

Studying PRKAB1 in primary tissues presents distinct challenges and opportunities compared to cell line models:

Sample preparation optimization:
For tissue lysate preparation, consider tissue-specific extraction buffers. While cell lines typically require 40μg of protein for Western blot detection of PRKAB1, primary tissues may require adjustment based on endogenous expression levels. For immunohistochemistry, enzyme-based antigen retrieval has proven effective for PRKAB1 detection and should be optimized for each tissue type .

Expression heterogeneity considerations:
Primary tissues exhibit cellular heterogeneity that can confound PRKAB1 analysis. Consider:

• Single-cell approaches or laser capture microdissection for cell-type specific analysis

• Co-staining with cell type-specific markers in immunohistochemistry

• Tissue microdissection prior to biochemical analysis

Phosphorylation stability differences:
PRKAB1 phosphorylation states in primary tissues are highly labile. From tissue extraction to analysis:

• Minimize time between sample collection and processing

• Use phosphatase inhibitor cocktails in all buffers

• Consider snap-freezing samples in liquid nitrogen immediately after collection

• For surgical specimens, record ischemia time as this affects AMPK activation

How can PRKAB1 antibodies be effectively utilized in AMPK complex purification strategies?

PRKAB1 antibodies offer strategic advantages in AMPK complex purification due to the subunit's central role in complex formation:

Immunoaffinity purification approach:
The monoclonal PRKAB1 antibody (1A7) has been validated for immunoprecipitation at dilutions of 1:50-200 , making it suitable for AMPK complex purification. This approach offers several advantages:

• Epitope consideration: Select antibodies that recognize epitopes outside the binding interfaces with α and γ subunits to maintain intact complexes. The clone 4H6H6 monoclonal antibody has been validated for this purpose .

• Elution strategy optimization: For functional studies of purified AMPK:

  • Gentle elution with excess immunizing peptide preserves activity

  • Low pH elution (pH 2.8) followed by immediate neutralization

  • For stringent purification, consider covalently coupling antibodies to beads to eliminate antibody contamination

• Sequential immunoprecipitation: To isolate specific AMPK complex compositions (e.g., α1β1γ1 versus α2β1γ1):

  • First round: Capture with PRKAB1 antibody

  • Elution under mild conditions

  • Second round: Capture with isoform-specific α subunit antibody

Tandem affinity purification design:
For higher purity, implement tandem affinity strategies:

• Express tagged PRKAB1 (e.g., FLAG-tagged) in cells of interest

• First purification step using anti-FLAG affinity

• Second purification using PRKAB1 antibody immunoprecipitation

• This approach yields highly pure AMPK complexes containing endogenous α and γ subunits

Sample preparation considerations:
When designing lysis conditions for AMPK complex purification:

• Use physiological salt concentrations (150 mM NaCl)

• Include 5-10% glycerol to stabilize protein interactions

• Add AMP (50-100 μM) to preserve active conformations

• Include both protease and phosphatase inhibitors

What are the emerging applications of PRKAB1 antibodies in studying subcellular compartmentalization of AMPK signaling?

Recent research has revealed sophisticated subcellular compartmentalization of AMPK signaling, with PRKAB1 antibodies emerging as crucial tools in this investigation:

Immunofluorescence co-localization studies:
PRKAB1 antibodies have been successfully used for immunofluorescence with enzyme antigen retrieval, enabling visualization of subcellular localization . For optimal results:

• High-resolution imaging approaches:

  • Super-resolution microscopy (STORM, PALM) to resolve nanoscale localization

  • Confocal microscopy with Airyscan for improved resolution within diffraction limit

  • Proximity ligation assay (PLA) to detect PRKAB1 interactions with compartment-specific proteins

• Compartment-specific markers for co-localization:

  • Plasma membrane: Na+/K+ ATPase, cadherin

  • Mitochondria: TOM20, MitoTracker

  • Lysosomal: LAMP1, LAMP2

  • Nuclear: Lamin B1, DAPI

  • Cytoskeletal: Tubulin, actin

Fractionation-based biochemical analysis:
When immunofluorescence results suggest compartmentalization, validate with biochemical fractionation:

• Optimized fractionation protocols:

  • Differential centrifugation for major organelles

  • Density gradient separation for membrane microdomains

  • Detergent-based separation of cytoskeletal-associated fractions

• Western blot analysis of fractions:

  • Probe with PRKAB1 antibodies (1:1000 dilution)

  • Include compartment-specific markers to verify fraction purity

  • Assess phosphorylation status to determine compartment-specific activation

Dynamic translocation analysis:
AMPK translocation between compartments is an emerging regulatory mechanism:

• Live-cell imaging approaches:

  • For endogenous PRKAB1: Immunofluorescence at defined time points

  • For exogenous studies: GFP-PRKAB1 fusions with careful validation against antibody-detected endogenous patterns

• Stimulus-dependent redistribution:

  • Energy stress (glucose deprivation, oligomycin)

  • Calcium flux (ionomycin, thapsigargin)

  • Hypoxia (CoCl₂, hypoxic chamber)

• Relationship between myristoylation and localization:

  • Compare wild-type versus G2A mutant localization

  • Use myristoylation inhibitors and monitor redistribution

  • Correlate with functional readouts of compartment-specific AMPK targets