Phospho-CD19 (Tyr531) Antibody

What is the function of CD19 and significance of Tyr531 phosphorylation?

CD19 functions as a coreceptor for the B-cell antigen receptor complex (BCR) on B-lymphocytes. It decreases the threshold for activation of downstream signaling pathways and triggers B-cell responses to antigens. CD19 activates pathways leading to phosphatidylinositol 3-kinase activation and mobilization of intracellular Ca²⁺ stores .

Tyr531 phosphorylation is particularly significant in B-cell signaling as it occurs extremely rapidly following B-cell activation. This phosphorylation is mediated by LYN kinase and occurs in a sequential pattern: Tyr531 phosphorylation happens first, followed by Tyr409, while Tyr500 phosphorylation appears more slowly and is more transient . This sequential phosphorylation pattern is critical for proper signal propagation through the BCR complex.

The molecular weight of phosphorylated CD19 protein detected in Western blot analysis is approximately 95 kDa . This information is essential for correctly identifying the target protein band. The apparent molecular weight may vary slightly depending on the cell type or tissue due to potential differences in post-translational modifications.

How does the phosphorylation kinetics of CD19 Tyr531 compare to other tyrosine residues in the signaling cascade?

The phosphorylation of CD19 tyrosine residues follows a specific temporal sequence after B-cell receptor activation. Tyr531 phosphorylation occurs extremely rapidly, representing one of the earliest events in the signaling cascade. This is followed by phosphorylation at Tyr409 . In contrast, phosphorylation of Tyr500 appears more slowly and is significantly more transient, quickly returning to basal levels .

This differential phosphorylation pattern suggests a hierarchical regulation mechanism where Tyr531 phosphorylation may serve as a priming event for subsequent phosphorylation of other tyrosine residues. Researchers investigating signaling kinetics should consider these temporal dynamics when designing time-course experiments to capture the complete signaling cascade.

What are the optimal normalization strategies when using CD19 (phospho Tyr531) cell-based ELISA?

For CD19 (phospho Tyr531) cell-based ELISA assays, multiple normalization strategies are recommended:

• GAPDH normalization: Anti-GAPDH antibody serves as an internal positive control for normalizing target absorbance values .

• Crystal Violet normalization: After the colorimetric measurement of HRP activity, Crystal Violet whole-cell staining determines cell density. This allows normalizing absorbance values to cell amounts, adjusting for plating differences .

• Total CD19 normalization: Using anti-CD19 antibody (non-phospho specific) to normalize the absorbance values of the phosphorylated target. This accounts for variations in total CD19 expression levels between samples .

The choice of normalization method should be based on experimental design and the specific research question. For comparing phosphorylation levels across different treatments, the total CD19 normalization approach provides the most accurate representation of relative phosphorylation status.

How can cross-reactivity with other phosphorylated proteins be assessed and minimized?

Assessing and minimizing cross-reactivity with other phosphorylated proteins is crucial for obtaining specific results when using Phospho-CD19 (Tyr531) antibodies. Several approaches can address this issue:

• Peptide competition assays: Treating the antibody with the antigen-specific peptide before Western blot analysis. As demonstrated in search result , the absence of signal in the lane treated with the antigen-specific peptide confirms antibody specificity.

• Validation in knockout/knockdown models: Testing the antibody in CD19-depleted cells to confirm the absence of signal.

• Dephosphorylation controls: Treating samples with phosphatases before analysis to verify that the signal depends on phosphorylation status.

• Antibody selection: Using affinity-purified antibodies that have been validated against the specific phosphorylation site. For example, the antibody described in result was "affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen."

What are the optimal sample preparation conditions for detecting phosphorylated CD19?

Optimal sample preparation for detecting phosphorylated CD19 (Tyr531) requires careful consideration of the following factors:

• Rapid sample processing: Phosphorylation states can change rapidly. Samples should be processed quickly and kept cold throughout preparation to preserve phosphorylation status.

• Phosphatase inhibitors: Include phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate) in all buffers to prevent dephosphorylation during sample preparation.

• Cell stimulation conditions: For B-cell activation studies, standardize stimulation conditions. According to the search results, serum stimulation has been successfully used to induce CD19 phosphorylation in COS7 cells .

• Sample types: The antibodies work with various sample types, including adherent cells and suspension cells , making them versatile for different experimental designs.

• Storage: Store prepared samples at -20°C or -80°C to prevent degradation and maintain phosphorylation status .

What controls should be included when studying CD19 Tyr531 phosphorylation in different cell types?

When studying CD19 Tyr531 phosphorylation across different cell types, several controls should be included:

• Positive controls: Include a cell type known to express CD19 and exhibit Tyr531 phosphorylation (e.g., B lymphocytes).

• Negative controls: Include a non-B cell type that does not express CD19.

• Stimulation controls: Include both stimulated and unstimulated samples to demonstrate induction of phosphorylation.

• Peptide competition controls: Include samples where the antibody is pre-incubated with the phospho-peptide immunogen to confirm signal specificity .

• Total CD19 detection: Use a non-phospho-specific CD19 antibody to normalize for total CD19 expression levels.

• Loading controls: Include housekeeping proteins (GAPDH, β-actin) for Western blot or ELISA normalization .

How does the choice of cell lysis buffer affect phospho-CD19 detection efficiency?

The choice of cell lysis buffer significantly impacts phospho-CD19 detection efficiency. Consider the following guidelines:

• Buffer composition: Use a buffer containing adequate detergent concentration (typically 1% NP-40 or Triton X-100) to solubilize membrane proteins like CD19 effectively.

• Phosphatase inhibitors: Include a comprehensive phosphatase inhibitor cocktail to preserve phosphorylation status:

  • Sodium orthovanadate (1-2 mM) for tyrosine phosphatases

  • Sodium fluoride (5-10 mM) for serine/threonine phosphatases

  • β-glycerophosphate (10 mM) for acid phosphatases

• Protease inhibitors: Include protease inhibitors to prevent protein degradation.

• Ionic strength: Moderate to high salt concentration (150-300 mM NaCl) helps maintain protein solubility while reducing non-specific interactions.

• pH consideration: Maintain pH between 7.2-7.5 to preserve phosphorylation status.

Most commercial antibody suppliers recommend using their optimized lysis buffers for best results, but the above guidelines can be used to develop custom buffers for specific experimental needs.

What are the common causes of false negative results when detecting Phospho-CD19 (Tyr531)?

Several factors can contribute to false negative results when detecting Phospho-CD19 (Tyr531):

• Rapid dephosphorylation: Tyr531 phosphorylation is extremely rapid but can also be transient. Insufficient phosphatase inhibition during sample preparation can lead to loss of signal.

• Improper sample handling: Extended processing time at room temperature can reduce phosphorylation signals.

• Suboptimal antibody dilution: Using too dilute antibody preparation. The recommended dilutions are 1:1000 for Western blotting and 1:100 for immunoprecipitation with Cell Signaling Technology's antibody , or 1:500-1:2000 for Western blotting with St John's Labs antibody .

• Inefficient protein transfer: Poor transfer efficiency during Western blotting can result in weak or absent signals.

• Blocking reagent incompatibility: Some blocking agents may interfere with phospho-epitope recognition.

• Cell stimulation conditions: Insufficient or inappropriate stimulation protocols may not induce CD19 phosphorylation effectively.

• Storage conditions: Improper storage of antibody (repeated freeze-thaw cycles) can degrade antibody quality and reduce sensitivity .

How can phosphorylation-specific signals be distinguished from non-specific binding?

Distinguishing phosphorylation-specific signals from non-specific binding requires several validation approaches:

• Peptide competition assays: Pre-incubate the antibody with the phospho-peptide immunogen. As shown in result , this should eliminate specific signals while non-specific signals remain.

• Phosphatase treatment controls: Treat duplicate samples with λ-phosphatase before analysis. Phosphorylation-specific signals should disappear after treatment.

• Multiple detection methods: Confirm findings using complementary techniques (e.g., verify Western blot results with ELISA or immunoprecipitation).

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of CD19 (approximately 95 kDa) .

• Signal induction: Verify that the signal increases with treatments known to induce CD19 phosphorylation (e.g., B-cell receptor stimulation).

• Knockout/knockdown validation: Test the antibody in CD19-deficient cells to confirm signal specificity.

What are the critical parameters for optimizing Western blot protocol for Phospho-CD19 (Tyr531) detection?

Optimizing Western blot protocols for Phospho-CD19 (Tyr531) detection requires attention to several critical parameters:

• Sample preparation:

  • Maintain samples at 4°C during preparation

  • Include comprehensive phosphatase inhibitor cocktail

  • Use optimized lysis buffer with appropriate detergent concentration

• Gel electrophoresis:

  • Use a gel percentage that provides good resolution around 95 kDa (typically 8-10% acrylamide)

  • Load adequate protein amount (typically 20-50 μg total protein)

• Transfer conditions:

  • Select appropriate membrane (PVDF typically provides better results for phospho-epitopes)

  • Optimize transfer time and voltage for high molecular weight proteins

• Blocking conditions:

  • Use BSA-based blocking solution rather than milk (milk contains phosphoproteins that may increase background)

  • Limit blocking time to prevent epitope masking

• Antibody incubation:

  • Use recommended dilution (1:1000-1:2000 for most antibodies)

  • Incubate at 4°C overnight for optimal sensitivity

  • Include Tween-20 (0.05-0.1%) in antibody dilution buffer

• Washing stringency:

  • Perform thorough washing steps with TBS-T to reduce background

  • Maintain consistent washing times and agitation

• Detection system:

  • Use enhanced chemiluminescence (ECL) or fluorescence-based detection systems

  • Optimize exposure time to prevent signal saturation

How can Phospho-CD19 (Tyr531) antibodies be utilized in studying B-cell receptor signaling pathways?

Phospho-CD19 (Tyr531) antibodies serve as valuable tools for investigating B-cell receptor (BCR) signaling pathways:

• Temporal signaling dynamics: Track the kinetics of CD19 phosphorylation at Tyr531 following BCR stimulation to map early signaling events.

• Signaling crosstalk: Investigate how CD19 Tyr531 phosphorylation influences or is influenced by other signaling molecules within the BCR complex.

• Functional studies: Correlate CD19 Tyr531 phosphorylation levels with downstream functional outcomes, such as calcium mobilization or activation of transcription factors.

• Drug discovery: Evaluate how potential therapeutic compounds affect CD19 phosphorylation as part of the BCR signaling pathway.

• Disease models: Compare CD19 Tyr531 phosphorylation patterns in normal B cells versus those from disease models or patient samples.

The antibodies can be employed in various techniques including Western blotting, immunoprecipitation, and ELISA , allowing for comprehensive analysis of phosphorylation status across different experimental settings.

What are the considerations for multiplex analysis of CD19 phosphorylation with other B-cell markers?

When performing multiplex analysis of CD19 phosphorylation alongside other B-cell markers, researchers should consider:

• Antibody compatibility: Ensure that antibodies against different targets do not interfere with each other's binding. This is particularly important for co-immunoprecipitation or co-immunofluorescence studies.

• Sequential vs. simultaneous detection: For Western blotting, determine whether sequential probing (stripping and reprobing) or simultaneous detection (using antibodies from different species) is more appropriate.

• Normalization strategies: When measuring multiple phosphorylation sites, normalize each to its respective total protein level rather than to a single housekeeping protein.

• Cell-based ELISA approach: The cell-based ELISA format allows for parallel detection of phospho-CD19, total CD19, and normalization controls like GAPDH .

• Species cross-reactivity: Consider the species reactivity of all antibodies in the multiplex panel. The Phospho-CD19 (Tyr531) antibodies available show reactivity with human, mouse, and in some cases, monkey samples .

• Signal separation: For fluorescence-based detection methods, ensure adequate spectral separation between fluorophores to prevent bleed-through in multicolor analysis.

What emerging technologies are enhancing the study of CD19 phosphorylation dynamics?

Several emerging technologies are advancing our ability to study CD19 phosphorylation dynamics:

• Mass spectrometry-based phosphoproteomics: Allows for unbiased, comprehensive analysis of phosphorylation sites and their stoichiometry.

• Single-cell phospho-flow cytometry: Enables analysis of phosphorylation events at the single-cell level, revealing population heterogeneity.

• CRISPR-based phosphorylation site mutants: Facilitates precise genetic manipulation to study the functional consequences of specific phosphorylation events.

• Biosensors and FRET-based approaches: Allow for real-time monitoring of phosphorylation events in living cells.

• Cell-based ELISA techniques: As described in result , these provide quantitative measurement of phosphorylation levels normalized to total protein expression.

• Phospho-specific nanobodies: Offer improved access to epitopes and potentially greater specificity for phosphorylated residues.

These technologies complement traditional antibody-based detection methods and are expanding our understanding of the dynamic regulation of CD19 phosphorylation in B-cell biology and related pathologies.

How do advances in antibody development impact the specificity and sensitivity of Phospho-CD19 (Tyr531) detection?

Recent advances in antibody development have significantly enhanced the specificity and sensitivity of Phospho-CD19 (Tyr531) detection:

• Recombinant antibody technology: Provides consistent lot-to-lot reproducibility compared to traditional polyclonal antibodies.

• Affinity purification techniques: As noted in result , antibodies "affinity-purified from rabbit antiserum by affinity-chromatography using epitope-specific immunogen" offer improved specificity.

• Monoclonal antibody development: Enables selection of clones with optimal specificity for the phosphorylated epitope.

• Rabbit monoclonal antibodies: Often provide superior affinity and specificity compared to mouse monoclonals, particularly for phospho-epitopes.

• Validation standards: More rigorous validation protocols, including knockout controls and phosphatase treatments, ensure antibody specificity.

• Application-specific optimization: Antibodies are increasingly optimized for specific applications (Western blot, ELISA, immunoprecipitation) with corresponding validated protocols.