CD19 (Ab-531) Antibody

What is CD19 (Ab-531) Antibody and what epitope does it recognize?

CD19 (Ab-531) Antibody (Product Code: CSB-PA253616) is a rabbit polyclonal antibody specifically targeting human CD19 protein. It was developed against a synthesized non-phosphopeptide derived from human CD19 around the tyrosine 531 phosphorylation site (D-S-Y(p)-E-N) . This antibody detects endogenous levels of total CD19 protein and is particularly valuable for studying CD19 signaling pathways where Y531 phosphorylation plays a critical role .

The antibody has been purified from rabbit antiserum using affinity chromatography with epitope-specific immunogen, ensuring high specificity for the target region . Western blot validation shows clear detection of CD19 in cells such as COS7 cells treated with serum (10%, 30 mins) .

What applications is CD19 (Ab-531) Antibody validated for?

CD19 (Ab-531) Antibody has been validated for the following experimental applications:

The antibody performs effectively in detecting endogenous CD19 protein in both human and mouse samples . It is particularly useful for studying CD19 in the context of B-cell receptor (BCR) signaling pathways .

How should CD19 (Ab-531) Antibody be stored for optimal stability?

For optimal stability and performance, CD19 (Ab-531) Antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can degrade antibody quality and compromise experimental results .

The antibody is provided in a stabilizing buffer consisting of rabbit IgG in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation helps maintain antibody integrity during storage.

Methodological approach: Aliquot the antibody upon first thaw to minimize freeze-thaw cycles. For working dilutions, store at 4°C for short-term use (1-2 weeks maximum), and return the stock to -20°C or -80°C for long-term storage.

What controls should be implemented when using CD19 (Ab-531) Antibody?

When designing experiments with CD19 (Ab-531) Antibody, several controls should be considered to ensure validity and reproducibility:

• Positive control: Use cell lines known to express CD19, such as COS7 cells treated with serum (10%, 30mins) which have been validated for this antibody .

• Negative control: Include CD19-negative cells or tissues to confirm specificity.

• Isotype control: Use rabbit IgG at the same concentration to assess non-specific binding.

• Peptide competition: For critical experiments, pre-incubate the antibody with the immunizing peptide to demonstrate binding specificity.

• Phosphorylation studies: When analyzing CD19-Y531 phosphorylation, include samples treated with phosphatase inhibitors and compare with untreated samples .

This methodological approach ensures reliable interpretation of results and helps distinguish between specific and non-specific signals, particularly important when studying CD19 phosphorylation at Y531 in BCR signaling pathways .

What are the optimal protocols for studying CD19-Y531 phosphorylation with this antibody?

CD19-Y531 phosphorylation is a critical event in B-cell signaling. To effectively study this phenomenon with CD19 (Ab-531) Antibody:

• Cell treatment protocol:

  • Stimulate B cells with anti-BCR antibody (1-5 minutes) to induce rapid CD19-Y531 phosphorylation

  • Include time points at 1, 5, and 15 minutes to capture the phosphorylation kinetics

  • Research shows phosphorylation typically peaks at 1 minute, decreases at 5 minutes, and returns to baseline by 15 minutes

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride)

  • Maintain samples at 4°C throughout processing

  • Use SDS-PAGE with 8-10% gels for optimal protein separation

• Immunoblotting conditions:

  • Transfer to PVDF membrane at 100V for 1 hour

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

  • Incubate with CD19 (Ab-531) Antibody at 1:1000 dilution overnight at 4°C

Research findings demonstrate that CD19-Y531 phosphorylation is significantly reduced when CD19 is co-engaged with CD47, highlighting the regulatory mechanisms in BCR signaling .

How does CD19 (Ab-531) Antibody perform in different subcellular fractionation experiments?

CD19 (Ab-531) Antibody can be effectively used for subcellular localization studies, particularly for investigating CD19 association with lipid rafts and BCR domains:

• Membrane fractionation protocol:

  • Use sucrose gradient ultracentrifugation to isolate lipid raft fractions

  • Solubilize cells in 1% Triton X-100 buffer at 4°C

  • Layer lysate on 5-40% sucrose gradient and centrifuge at 200,000g for 16 hours

  • Collect fractions and analyze by immunoblotting with CD19 (Ab-531) Antibody at 1:500 dilution

• Interpretation considerations:

  • CD19 normally associates with BCR clusters in lipid rafts to enhance signaling

  • Research demonstrates that CD47xCD19 co-engagement prevents CD19 from migrating to BCR domains, thereby impairing signaling

  • Compare fractionation patterns before and after BCR stimulation to assess CD19 translocation

This methodological approach helps researchers understand how CD19 trafficking influences BCR signaling thresholds, providing insights into B-cell activation mechanisms .

How can CD19 (Ab-531) Antibody be integrated into CAR-T cell research protocols?

CD19 (Ab-531) Antibody offers valuable applications in chimeric antigen receptor T-cell (CAR-T) research, particularly for monitoring CD19 expression and antigen density:

• Assessment of target antigen density:

  • CD19 antigen density can be quantified using the antibody with calibration beads for flow cytometry

  • Research indicates CAR-T cell activity is highly dependent on CD19 antigen density

  • CD19–CD28ζ CAR-T cells show different efficiency in targeting CD19-low cells compared to CD19-4-1BBζ CAR-T cells

• Monitoring CD19 expression pre/post-therapy:

  • Use CD19 (Ab-531) Antibody (1:500 dilution) for Western blot analysis of patient samples

  • Compare CD19 expression levels before and after treatment

  • This approach helps identify antigen escape mechanisms, including epitope loss and defective cell surface trafficking of CD19

• Protocol for analyzing relapse samples:

  • Process bone marrow or lymph node biopsies within 24 hours

  • Prepare single-cell suspensions and lyse for protein extraction

  • Run Western blot with CD19 (Ab-531) Antibody and analyze band intensity to quantify CD19 levels

Research findings suggest that CD19 expression may be maintained after non-CAR-T CD19-directed therapy, and some patients with disease relapse after CAR-T may benefit from subsequent CD19-directed therapy .

What are the methodological considerations for using CD19 (Ab-531) Antibody in CD19-TLR9 signaling studies?

Recent research has identified a crucial role of CD19 in controlling TLR9 responses in human B cells. When using CD19 (Ab-531) Antibody to study this pathway:

• Experimental setup for phosphorylation analysis:

  • Stimulate B cells with CpG oligodeoxynucleotides (2 μM) to activate TLR9

  • Include parallel samples with BCR crosslinking for comparison

  • Collect cells at 5-30 minute intervals post-stimulation

  • Process for Western blot with phospho-specific antibodies for BTK (Y551) and AKT (S473)

  • Use CD19 (Ab-531) Antibody (1:1000) to confirm CD19 expression levels

• CD19 knockdown validation protocol:

  • When using CD19 knockdown cell lines (e.g., Ramos B-cells with CD19 shRNA)

  • Confirm knockdown efficiency using CD19 (Ab-531) Antibody

  • Compare phosphorylation of downstream targets (BTK, AKT) between control and knockdown cells

  • Research shows AKT phosphorylation at S473 is strongly reduced in CD19-deficient cells after CpG stimulation

This methodological approach helps elucidate how CD19 contributes to TLR9-mediated signaling in B cells, which is important for understanding B-cell responses to both self and foreign DNA .

How can CD19 (Ab-531) Antibody be utilized in studying CD19-positive antibody-secreting cells?

CD19 (Ab-531) Antibody can be valuable for investigating the recently discovered CD19-positive antibody-secreting cells (ASCs) that provide immune memory:

• Protocol for identifying CD19+ ASCs:

  • Process bone marrow samples to isolate mononuclear cells

  • Perform flow cytometry staining for plasma cell markers (CD138) alongside CD19

  • Sort CD19+ and CD19- ASC populations for comparative analysis

  • Use CD19 (Ab-531) Antibody in Western blot (1:1000) to validate CD19 expression in sorted populations

• Functional analysis considerations:

  • Compare antibody secretion capacity between CD19+ and CD19- ASCs using ELISPOT

  • Analyze gene expression profiles of both populations

  • Research indicates that both CD19+ and CD19- ASCs share a developmental pathway and are capable of providing long-lasting immune memory

• Methodological advantages:

  • The antibody's specificity for total CD19 enables detection across differentiation stages

  • Western blot analysis can reveal potential variations in CD19 expression levels

These approaches provide insights into the heterogeneity of memory ASCs and their role in maintaining humoral immunity through continued production of antibodies specific for previously encountered pathogens or vaccine antigens .

How can nonspecific binding be reduced when using CD19 (Ab-531) Antibody in Western blot?

Nonspecific binding is a common challenge when working with polyclonal antibodies like CD19 (Ab-531) Antibody. To minimize this issue:

• Optimized blocking protocol:

  • Use 5% BSA in TBST rather than milk for blocking (2 hours at room temperature)

  • For particularly problematic samples, add 0.1% Tween-20 to the blocking buffer

  • Consider using commercial blocking reagents specifically designed for phospho-antibodies

• Antibody dilution optimization:

  • Begin with the manufacturer's recommended range (1:500-1:3000)

  • Perform a dilution series to determine optimal concentration for your specific samples

  • Higher dilutions (1:2000-1:3000) often reduce background while maintaining specific signal

• Washing procedure enhancement:

  • Implement 5 washes (5 minutes each) with TBST between antibody incubations

  • Use fresh, cold washing buffer for each wash

  • Consider adding 0.5M NaCl to washing buffer for one of the washes to disrupt low-affinity interactions

These methodological refinements significantly improve signal-to-noise ratio when working with CD19 (Ab-531) Antibody, as demonstrated in studies examining CD19 phosphorylation in COS7 cells .

What strategies address inconsistent results when detecting CD19 in different sample types?

Researchers may encounter variability when detecting CD19 across different sample types. To address this challenge:

• Sample-specific optimization protocol:

  • For cell lines: Lyse 1-2 × 10⁶ cells in 100 μl RIPA buffer supplemented with protease inhibitors

  • For primary B cells: Use 5-10 × 10⁶ cells with gentler NP-40 lysis buffer

  • For tissue samples: Homogenize in buffer containing 1% Triton X-100, 150 mM NaCl, 20 mM Tris

  • Include phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄) for all samples when studying phosphorylation

• Protein loading considerations:

  • Load 20-50 μg total protein for cell lines

  • Increase to 50-80 μg for primary samples

  • Always normalize to loading controls (β-actin, GAPDH)

• Sample handling protocol:

  • Process all samples on ice to prevent protein degradation

  • Add 1 mM PMSF (fresh) to lysis buffer immediately before use

  • Avoid repeated freeze-thaw cycles of protein lysates

Research has shown that CD19 detection can be particularly challenging in samples with low B-cell numbers or when studying CD19 internalization (up to 20% after 4 hours with bivalent anti-CD19 mAb) .

How should CD19 phosphorylation signaling data be analyzed in BCR activation studies?

Proper interpretation of CD19 phosphorylation data is critical for understanding its role in BCR signaling:

• Phosphorylation kinetics analysis protocol:

  • Quantify band intensity using densitometry software

  • Normalize phospho-CD19 (Y531) to total CD19 expression

  • Plot time-course data to visualize phosphorylation dynamics

  • Research shows CD19-Y531 phosphorylation typically peaks at 1 minute after BCR stimulation, decreases at 5 minutes, and returns to baseline by 15 minutes

• Comparative analysis framework:

  • When comparing different experimental conditions (e.g., CD19 monovalent vs. bivalent engagement):

  • Calculate fold change in phosphorylation relative to unstimulated control

  • Use statistical tests (paired t-test) to determine significance

  • Consider area under the curve analysis for comprehensive phosphorylation response

• Interpretation guidelines:

  • Reduced CD19-Y531 phosphorylation indicates impaired BCR signaling

  • Changes in phosphorylation pattern (timing, intensity) reflect alterations in signal transduction

  • Research demonstrates that CD47xCD19 co-engagement significantly decreases CD19-Y531 phosphorylation compared to control conditions

This methodological approach provides insights into how different molecular interactions influence BCR signaling thresholds, which is crucial for understanding B-cell activation in both normal and pathological conditions .

What considerations are important when comparing CD19 (Ab-531) Antibody results with other CD19-directed research tools?

When integrating results from CD19 (Ab-531) Antibody with other CD19-directed research tools (such as CAR-T cells or monoclonal antibodies):

• Epitope mapping considerations:

  • CD19 (Ab-531) Antibody targets the region around Y531

  • Many therapeutic anti-CD19 antibodies and CARs target different epitopes

  • Humanized CD19 scFv derived from mouse CD19 FMC63 clone has been shown to specifically target human CD19 with no detectable off-target binding

  • Differences in targeting epitopes may explain varied experimental outcomes

• Functional effect comparison protocol:

  • When comparing anti-CD19 mAb with CD19-CAR:

  • Document concentration/dose equivalence

  • Measure cytokine production, proliferation, and cytotoxicity

  • Research shows anti-CD19-CAR-transduced T cells have superior antilymphoma efficacy compared with the anti-CD19 monoclonal antibody from which the anti-CD19 CAR was derived

• Data integration framework:

  • Create comparison tables documenting:

    • Epitope targeted

    • Binding affinity

    • Functional outcomes

  • Acknowledge that different CD19-targeting approaches may yield complementary insights

  • Consider sequence of CD19-directed therapies as this may impact antigen expression and escape mechanisms

This methodological approach enables researchers to construct a more comprehensive understanding of CD19 biology by integrating findings from diverse experimental tools .