Phospho-Bad (Ser136) Antibody

What is the significance of Bad phosphorylation at Serine 136?

Phosphorylation of Bad at Serine 136 is a critical regulatory mechanism in cell survival signaling. When Bad is phosphorylated at this residue, primarily by Akt kinase in the PI3K/Akt pathway, it promotes cell survival by preventing Bad from binding to anti-apoptotic proteins such as Bcl-XL and Bcl-2. This phosphorylation facilitates the binding of Bad to 14-3-3 proteins, sequestering it in the cytosol and inhibiting its pro-apoptotic function . Understanding this phosphorylation state provides valuable insights into the balance between cell survival and apoptotic pathways in various physiological and pathological conditions .

What are the common applications for Phospho-Bad (Ser136) antibodies?

Phospho-Bad (Ser136) antibodies are utilized across multiple experimental platforms:

• Western Blotting (WB): For detecting and quantifying phosphorylated Bad in cell or tissue lysates

• Immunoprecipitation (IP): For isolating phospho-Bad protein complexes

• ELISA: For quantitative measurement of phospho-Bad levels

• Immunohistochemistry (IHC): For localizing phospho-Bad in tissue sections

• Immunofluorescence (IF): For visualizing subcellular localization of phospho-Bad

Each application requires specific optimization parameters including antibody dilution, incubation time, and detection methods as detailed in the product-specific protocols .

How should I design control experiments when studying Bad phosphorylation?

Robust control experiments are essential when studying Bad phosphorylation:

Positive controls:

• Use lysates from cells treated with growth factors or other activators of the PI3K/Akt pathway

• For FACE assays, include wells probed with total-Bad antibody to normalize phosphorylation levels

Negative controls:

• Include lysates from cells treated with PI3K inhibitors (e.g., LY294002) which should reduce phosphorylation at Ser136

• For immunoassays, include secondary antibody-only controls to assess non-specific binding

Treatment validation:

• Verify pathway activation by also detecting phospho-Akt (Ser473), which is upstream of Bad phosphorylation

• Include time course experiments to capture dynamic phosphorylation changes

What are the optimal sample preparation methods for detecting phospho-Bad (Ser136)?

Proper sample preparation is critical for maintaining phosphorylation status:

• For cell lysis:

  • Use a lysis buffer containing phosphatase inhibitors to prevent dephosphorylation

  • Maintain cold temperatures throughout sample processing

  • Avoid ionic detergents like SDS that may denature capture antibodies in immunoassays

• For tissue samples:

  • Flash freeze tissues immediately after collection

  • Homogenize in phosphatase inhibitor-containing buffer

  • Consider phospho-enrichment techniques for low abundance samples

• For fixation in cell-based assays:

  • Use 4% formaldehyde in PBS for adherent cells

  • Fix cells at approximately 80% confluence

  • Storage of fixed cells should be at refrigerated temperatures in sealed containers

How do I properly normalize phospho-Bad (Ser136) signals in western blots and cell-based assays?

Accurate normalization is essential for quantitative analysis:

For Western blotting:

• Run parallel blots or strip and reprobe for total Bad protein

• Additionally normalize to a stable housekeeping protein (e.g., β-actin, GAPDH)

• Calculate the ratio of phospho-Bad to total Bad to determine the proportion of phosphorylated protein

For cell-based assays (e.g., FACE):

• Use duplicate plates where one is probed for phospho-Bad and the other for total Bad

• Incorporate Crystal Violet staining to normalize for cell number variations between wells

• Calculate phospho-Bad/total Bad ratios after normalizing each for cell number

What could cause inconsistent or weak phospho-Bad (Ser136) signals?

Several factors can affect signal detection:

Common issues and solutions:

• Rapid dephosphorylation: Ensure phosphatase inhibitors are fresh and active in all buffers

• Low endogenous levels: Some antibodies may only detect transfected levels of phospho-Bad

• Cross-reactivity: Verify antibody specificity, particularly in multi-species studies

• Signal variability: Use matched antibody pairs from the same manufacturer for quantitative assays

• Storage-related degradation: Aliquot antibodies to avoid freeze/thaw cycles; store at recommended temperatures (-20°C)

How can phospho-Bad (Ser136) be used as a biomarker for studying cell fate decisions in cerebral ischemia?

Phospho-Bad (Ser136) serves as an integrated checkpoint between survival and death signaling in neuronal cells:

Research has demonstrated that after transient focal cerebral ischemia (tFCI), Bad acts as a converging signaling molecule between the survival PI3-K/Akt pathway and the death JNK pathway. In the peripheral area of cerebral ischemia, Akt activity increases at 3 hours post-injury, leading to increased phospho-Bad (Ser136) expression. This survival signal is later overwhelmed by increasing JNK activity (7-24 hours post-injury), which correlates with decreased phospho-Bad (Ser136) and increased Bad/Bcl-XL dimerization .

Experimental approaches for studying this transition include:

• Time course analysis of phospho-Bad (Ser136) levels after ischemic insult

• Co-immunoprecipitation of Bad with 14-3-3 versus Bcl-XL or Bcl-2 to track dimerization patterns

• Pharmacological manipulation with pathway inhibitors (LY294002 for PI3K/Akt; SP600125 for JNK)

• Co-localization studies of phospho-Bad (Ser136) with phospho-Akt (Ser473)

What techniques allow for quantitative measurement of Bad phosphorylation dynamics in living cells?

Several advanced techniques enable dynamic monitoring of Bad phosphorylation:

FACE™ (Fast Activated Cell-based ELISA):

• Allows measurement of phospho-Bad relative to cell number or total Bad protein

• Can be performed in 96-well format for high-throughput screening

• Provides a fixed cell record that can be stored for weeks before analysis

Multiplexed electrochemiluminescence assays:

• Systems like MSD platforms allow simultaneous detection of multiple phosphorylation sites

• Provide high sensitivity with broad dynamic range

• Enable comparison of multiple pathway components in a single sample

Phospho-specific antibody pairs for ELISA:

• Matched antibody pairs allow customization for specific research needs

• Compatible with multiple detection technologies including fluorophores, lanthanides, and biotin

• Can be adapted to various platforms such as MSD, Quanterix Simoa, and Alpha Technology

How does the stoichiometry of Bad phosphorylation at different sites (Ser112, Ser136, Ser155) affect its function?

Bad activity is regulated through a complex pattern of multi-site phosphorylation:

While phosphorylation at Ser136 is primarily mediated by Akt kinase, Bad is also phosphorylated at Ser112 by p90RSK and mitochondria-anchored PKA, and at Ser155 by PKA. These phosphorylation events work cooperatively but have distinct molecular consequences:

• Phosphorylation at Ser136 and Ser112 promotes heterodimerization with 14-3-3 proteins

• This interaction facilitates subsequent phosphorylation at Ser155 within the BH3 domain

• Phosphorylation at Ser155 is particularly critical as it directly blocks the dimerization of Bad with Bcl-XL

Research approaches to study these relationships include:

• Site-directed mutagenesis to create phospho-mimetic or phospho-deficient Bad variants

• Time-resolved phosphorylation analysis using phospho-specific antibodies against each site

• Structural studies of Bad interaction with partner proteins under different phosphorylation states

• Cell-based assays comparing consequences of selective kinase inhibition

What are the recommended validation steps when using a new batch of phospho-Bad (Ser136) antibody?

Thorough validation ensures consistent experimental results:

How should different types of phospho-Bad (Ser136) antibodies be selected for specific applications?

Selection criteria depend on experimental requirements:

Monoclonal vs. Polyclonal considerations:

• Monoclonal antibodies (e.g., D25H8) provide high specificity and lot-to-lot consistency

• Polyclonal antibodies may offer higher sensitivity for some applications

Matched antibody pairs:

• Essential for quantitative assays where both phosphorylated and total protein must be measured

• Ensure both antibodies have similar affinities and performance characteristics

Species reactivity:

• Verify cross-reactivity with the species being studied

• Some antibodies may have limited reactivity (e.g., mouse-only) or require validation for predicted reactivity based on sequence homology

Recombinant vs. traditional antibodies:

• Recombinant antibodies offer superior lot-to-lot consistency and continuous supply

• Consider for long-term studies where antibody consistency is critical