PRKACB Antibody, FITC conjugated

What is PRKACB and what cellular pathways does it regulate?

PRKACB, also known as PKA C-beta, belongs to the protein kinase superfamily, specifically within the AGC Ser/Thr protein kinase family and cAMP subfamily. It functions as a catalytic subunit of cAMP-dependent protein kinase (PKA), mediating signaling triggered by receptor binding to G-protein coupled receptors (GPCRs) . When cellular cAMP levels increase, inactive PKA holoenzyme (composed of two regulatory and two catalytic subunits) dissociates, releasing monomeric catalytic subunits that can phosphorylate various target proteins . Through this mechanism, PRKACB participates in regulating diverse cellular processes including cell proliferation, cell cycle progression, differentiation, microtubule dynamics, and nuclear envelope dynamics .

What are the molecular characteristics of PRKACB antibodies?

PRKACB antibodies typically target epitopes specific to the protein kinase cAMP-dependent catalytic beta subunit. According to product data, commercially available PRKACB antibodies have the following characteristics:

The FITC-conjugated variants maintain the specificity of unconjugated antibodies while providing direct fluorescent detection capability through the conjugated fluorescein isothiocyanate molecule .

What applications are FITC-conjugated PRKACB antibodies validated for?

FITC-conjugated PRKACB antibodies are primarily designed for immunofluorescence applications, allowing direct visualization of PRKACB in fixed cells and tissues without requiring secondary antibody incubation . Based on the available information, these antibodies are particularly suitable for:

• Immunofluorescence (IF)

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Immunocytochemistry (ICC)

While unconjugated PRKACB antibodies are extensively validated for Western blot applications with at least 21 published references, the FITC-conjugated versions are optimized specifically for microscopy-based applications .

What are the recommended dilutions and protocols for FITC-conjugated PRKACB antibodies?

Optimal dilutions for FITC-conjugated PRKACB antibodies vary depending on the specific application and experimental conditions. Based on the available data for similar antibodies:

It is strongly advised that researchers titrate the antibody in their specific experimental systems to determine optimal concentration . The fluorescent signal may vary depending on tissue type, fixation method, and antigen retrieval protocol employed.

What antigen retrieval methods are recommended for PRKACB antibody applications?

For optimal results in immunohistochemistry and immunofluorescence applications with PRKACB antibodies, the following antigen retrieval methods are recommended:

• Primary suggestion: TE buffer (Tris-EDTA) at pH 9.0

• Alternative method: Citrate buffer at pH 6.0

The choice between these methods may depend on the specific tissue type and fixation protocol used. For highly cross-linked tissues or those fixed for extended periods, the higher pH TE buffer method may provide superior epitope exposure.

What are the optimal storage conditions for preserving FITC-conjugated PRKACB antibody activity?

To maintain optimal activity of FITC-conjugated PRKACB antibodies, the following storage conditions are recommended:

• Temperature: Store at -20°C

• Buffer: PBS containing 0.02% sodium azide and 50% glycerol, pH 7.3

• Stability: Stable for one year after shipment when properly stored

• Aliquoting: Generally unnecessary for -20°C storage of small volumes, though may be advisable for larger volumes to prevent freeze-thaw cycles

It's important to note that FITC conjugates are light-sensitive, so antibodies should be protected from prolonged light exposure during storage and handling to prevent photobleaching of the fluorophore.

How can FITC-conjugated PRKACB antibodies be used to study cAMP signaling dynamics?

FITC-conjugated PRKACB antibodies provide a valuable tool for studying the spatial and temporal dynamics of cAMP-dependent protein kinase activation and localization. These antibodies can be used to:

• Visualize the subcellular distribution of PRKACB in response to stimuli that elevate cAMP levels

• Examine translocation of catalytic subunits between cellular compartments following PKA activation

• Investigate co-localization with PKA regulatory subunits or downstream substrates using multi-channel fluorescence microscopy

The direct conjugation to FITC eliminates potential signal amplification artifacts that can occur with secondary antibody detection systems, allowing for more quantitative assessment of PRKACB distribution .

How can researchers distinguish between PRKACA and PRKACB in experimental systems?

Distinguishing between the highly homologous catalytic subunits PRKACA (alpha) and PRKACB (beta) requires careful antibody selection and experimental controls:

• Use antibodies raised against unique epitopes specific to each isoform

• Validate antibody specificity using knockout or knockdown controls

• Consider the expression pattern differences - some tissues preferentially express one isoform over the other

• For phosphorylation studies, note that regulatory phosphorylation sites like Thr198 may be conserved between both isoforms

In cases where absolute specificity is required, complementary techniques such as RT-qPCR for isoform-specific mRNA detection may be necessary to confirm antibody-based findings.

What considerations are important when studying DNAJB1-PRKACA fusion in fibrolamellar carcinoma research?

The DNAJB1-PRKACA fusion protein is a critical oncogenic driver in fibrolamellar carcinoma (FLC). When using PRKACB antibodies in research related to this fusion, several important considerations apply:

• Standard PRKACA antibodies may recognize both wild-type PRKACA and the DNAJB1-PRKACA fusion protein, potentially complicating interpretation

• PRKACB antibodies provide a valuable control to distinguish PRKACA-specific effects from general PKA catalytic subunit effects

• The fusion breakpoint creates unique neoantigens that can be specifically recognized by T cells, with peptides like EIFDRYGEEV being presented on HLA-A*68:02

• PRKACB expression and activity may be altered in FLC as a compensatory mechanism for altered PRKACA function

These fusion proteins generate neoantigens that have potential as immunotherapy targets, with endogenous T-cell responses documented in FLC patients .

How can researchers optimize signal-to-noise ratio when using FITC-conjugated PRKACB antibodies?

Achieving optimal signal-to-noise ratio with FITC-conjugated antibodies requires attention to several technical factors:

• Fixation optimization: Overfixation can mask epitopes while underfixation may compromise cellular architecture

• Autofluorescence reduction: Tissues like brain contain lipofuscin that autofluoresces in the same spectrum as FITC; treatment with Sudan Black B can reduce this background

• Blocking optimization: Thorough blocking with appropriate sera (5-10% normal serum from the species of the secondary antibody) or BSA (3-5%) can minimize nonspecific binding

• Antibody concentration: Titrate antibody dilution to determine the optimal concentration that provides specific signal with minimal background

• Washing stringency: More frequent or longer washes with PBS containing 0.05-0.1% Tween-20 can reduce background without compromising specific signal

What controls should be included when using FITC-conjugated PRKACB antibodies?

Rigorous experimental design should include the following controls when using FITC-conjugated PRKACB antibodies:

• Isotype control: FITC-conjugated rabbit IgG at the same concentration to assess nonspecific binding

• Negative tissue control: Samples known to express low or undetectable levels of PRKACB

• Positive tissue control: Human brain or mouse brain tissue, which have confirmed PRKACB expression

• Absorption control: Pre-incubation of the antibody with excess immunizing peptide to confirm binding specificity

• Knockdown validation: Samples with PRKACB expression reduced through siRNA or CRISPR to confirm antibody specificity

For publications, it is advisable to include images of both positive and negative controls alongside experimental samples.

What are common technical issues when using FITC-conjugated antibodies and how can they be addressed?

When working with FITC-conjugated PRKACB antibodies, researchers may encounter several common technical challenges:

• Photobleaching: FITC is relatively prone to photobleaching

  • Solution: Minimize exposure to light during all handling steps, use anti-fade mounting media, and capture images promptly

• pH sensitivity: FITC fluorescence is optimal at alkaline pH (>7.0) and diminishes in acidic environments

  • Solution: Ensure all buffers, particularly mounting media, are maintained at pH 7.2-8.0

• Spectral overlap: FITC emission overlaps with other green fluorophores and some autofluorescence

  • Solution: Use appropriate filter sets and consider alternative conjugates (e.g., Alexa Fluor 488) for multi-color experiments

• Fixative compatibility: Some fixatives can affect FITC fluorescence intensity

  • Solution: Compare 4% paraformaldehyde, methanol, and acetone fixation to determine optimal protocol

How should researchers quantify and analyze data obtained with FITC-conjugated PRKACB antibodies?

For rigorous quantitative analysis of FITC-conjugated PRKACB antibody staining:

• Subcellular localization analysis: Use colocalization analysis with nuclear, cytoplasmic, or organelle markers to quantify PRKACB distribution

• Signal intensity quantification: Measure mean fluorescence intensity within defined cellular regions, normalizing to appropriate reference standards

• Population heterogeneity assessment: Apply single-cell analysis techniques to characterize cell-to-cell variation in PRKACB expression or localization

• Activation state determination: Combine with phospho-specific antibodies against PKA substrates to correlate PRKACB localization with signaling activity

These approaches provide more meaningful biological insights than simple presence/absence determinations of the target protein .

What are the considerations for interpreting results in the context of cAMP signaling pathways?

When interpreting PRKACB antibody staining results in the context of cAMP signaling:

• Consider that PRKACB is only one component of the PKA holoenzyme, which includes regulatory subunits that determine cAMP sensitivity and subcellular localization

• Recognize that PKA activity is spatially regulated through A-kinase anchoring proteins (AKAPs)

• Acknowledge that changes in PRKACB localization may reflect both altered expression and redistribution of existing protein

• Interpret results in the context of upstream cAMP-generating stimuli and downstream substrate phosphorylation

• Consider that cAMP signals are often compartmentalized within cells, resulting in localized PKA activation