PP2B12 Antibody

What is PP2B-B1/2 antibody and what cellular proteins does it target?

PP2B-B1/2 Antibody (such as the D-1 clone) is a mouse monoclonal IgG2b kappa light chain antibody that specifically detects PP2B-B1/2 protein of human origin. PP2B-B1/2 functions as a protein phosphatase, playing a crucial role in regulating cellular signaling pathways through dephosphorylation of serine and threonine residues. This activity is essential for maintaining cellular homeostasis, regulating cell division, and controlling apoptosis .

What detection methods are compatible with PP2B-B1/2 antibody?

PP2B-B1/2 antibody has been validated for multiple detection techniques including:

• Western blotting (WB)

• Immunoprecipitation (IP)

• Immunofluorescence (IF)

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

• Enzyme-linked immunosorbent assay (ELISA)

Success with these methods depends on proper antibody titration and validation in your specific experimental system.

What are the available conjugated forms of PP2B-B1/2 antibody?

How should I determine the optimal working dilution for PP2B-B1/2 antibody in my experiments?

For optimal results, the working dilution of PP2B-B1/2 antibody must be determined empirically for each application and experimental system. Generally, antigen affinity-purified polyclonal antibodies require lower dilutions (1.7-15 μg/mL) compared to monoclonal antibodies (5-25 μg/mL).

Methodology:

• Perform preliminary studies using a broad range of antibody concentrations

• For tissue sections, begin with overnight incubation at 4°C

• For cellular staining, start with 1-hour incubation at room temperature

• Maintain consistent incubation time and temperature when comparing different concentrations

• Select the concentration providing optimal signal-to-noise ratio

What controls should I include when using PP2B-B1/2 antibody for validating specificity?

Proper experimental controls are essential for antibody validation:

• Positive control: Use cell lines known to express the target (e.g., Raji cells for certain antibodies)

• Negative control: Include cell lines that don't express the target (e.g., K562 cells may serve as negative controls for some antibodies)

• Secondary antibody control: Samples treated with secondary antibody only

• Isotype control: Include an irrelevant antibody of the same isotype

• Transfected vs. non-transfected cells: Compare expression in cells transfected with the target protein against non-transfected cells

This comprehensive approach provides confidence in antibody specificity and minimizes false positive results.

How can I minimize cross-reactivity when using PP2B-B1/2 antibody in multi-protein detection systems?

To reduce cross-reactivity:

• Optimize blocking conditions using non-animal proteins (e.g., LiCor blocking buffer)

• Perform thorough washing steps between antibody incubations

• For multiplex detection, consider using primary antibodies from different host species

• Pre-adsorb antibodies with potential cross-reactive proteins

• Validate antibody specificity using knockout or knockdown models

• For immunoblotting, consider membrane stripping controls between different antibodies

• When using fluorescent detection systems, ensure proper filter settings to avoid spectral overlap

How can I use PP2B-B1/2 antibody to investigate protein-protein interactions in signaling pathways?

For investigating protein-protein interactions involving PP2B-B1/2:

• Co-immunoprecipitation (Co-IP):

  • Use PP2B-B1/2 antibody conjugated to agarose beads

  • Optimize lysis conditions to preserve protein complexes (consider mild detergents)

  • Include appropriate controls (IgG isotype control, input lysate)

  • Verify interactions by reciprocal Co-IP with antibodies against suspected binding partners

• Proximity Ligation Assay (PLA):

  • Combine PP2B-B1/2 antibody with antibodies against potential interacting proteins

  • Requires optimization of fixation and permeabilization protocols

  • Controls should include single antibody conditions and known non-interacting proteins

• FRET/BRET Analysis:

  • For live-cell interaction studies

  • May require fluorescent protein tagging of suspected interaction partners

  • Combine with antibody-based verification of interaction specificity

What approaches should I use to quantify relative levels of PP2B-B1/2 phosphatase activity in different cellular contexts?

For quantitative assessment of PP2B-B1/2 activity:

• Immunoblotting combined with phosphatase activity assays:

  • Immunoprecipitate PP2B-B1/2 using the specific antibody

  • Measure phosphatase activity using phosphorylated substrate peptides

  • Include phosphatase inhibitors as negative controls

  • Normalize activity to total PP2B-B1/2 protein levels determined by western blotting

• Live-cell phosphatase sensors:

  • Use FRET-based reporters for dynamic activity measurement

  • Complement with immunofluorescence using PP2B-B1/2 antibody for localization studies

• Substrate trapping approaches:

  • Combine with mass spectrometry to identify novel substrates

  • Verify using PP2B-B1/2 antibody in reverse immunoprecipitation experiments

How can epitope structure influence PP2B-B1/2 antibody specificity when analyzing protein complexes?

The epitope recognized by PP2B-B1/2 antibody can significantly impact experimental outcomes:

• Epitope masking in protein complexes:

  • Binding partners may obscure the epitope, reducing antibody accessibility

  • Consider multiple antibodies targeting different regions of PP2B-B1/2

  • Native vs. denatured conditions may yield different results

• Conformational epitopes:

  • Some antibodies recognize three-dimensional structures that may be lost in denatured samples

  • May require native conditions for immunoprecipitation experiments

• Cross-reactivity with structurally similar domains:

  • PP2B belongs to a family of protein phosphatases with conserved domains

  • Epitope mapping and specificity validation are crucial

  • Consider tests with recombinant family members to assess cross-reactivity

How can I distinguish between true positive signals and non-specific binding when using PP2B-B1/2 antibody?

To differentiate specific from non-specific signals:

• Perform titration experiments:

  • Compare signal intensity across different antibody concentrations

  • Specific signals typically show dose-dependent relationships

  • Non-specific binding may not diminish proportionally with dilution

• Peptide competition assays:

  • Pre-incubate antibody with excess target peptide

  • Specific signals should be blocked, while non-specific signals persist

• Multiple detection methods:

  • Confirm findings using alternative techniques (e.g., IF vs. WB)

  • Specific signals should be consistent across methodologies

• Cellular localization consistency:

  • Verify that subcellular localization matches known distribution patterns

  • Non-specific signals often show inconsistent localization patterns

Why might I observe inconsistent results between Western blotting and immunohistochemistry using PP2B-B1/2 antibody?

Discrepancies between Western blotting and immunohistochemistry can arise from:

• Epitope accessibility differences:

  • Denatured proteins in WB vs. partially preserved structures in IHC

  • Some epitopes may be accessible only in certain techniques

• Fixation effects:

  • Formalin fixation can mask epitopes through cross-linking

  • Antigen retrieval methods may be necessary for IHC but irrelevant for WB

• Context-dependent expression:

  • Cell-type specific expression in tissues may differ from whole tissue lysates

  • Consider single-cell techniques to resolve heterogeneity

• Protocol optimization requirements:

  • Each technique requires specific optimization

  • Buffer compositions, blocking reagents, and detection systems differ significantly

• Antibody clone suitability:

  • Some antibody clones perform better in certain applications

  • Consider application-specific validation data when selecting antibodies

What factors might contribute to false positive signals in immunohistochemistry when using PP2B-B1/2 antibody?

Several factors can generate false positive IHC signals:

• Endogenous peroxidase activity:

  • Inhibit using appropriate blocking reagents (e.g., hydrogen peroxide)

  • Critical when using HRP-conjugated detection systems

• Endogenous biotin:

  • Can interfere with biotin-streptavidin detection methods

  • Use biotin blocking systems or alternative detection methods

• Cross-reactivity with denatured class II HLA:

  • Some antibodies may react with denatured HLA at frequencies up to 11% in certain populations

  • Use proper controls and alternative detection methods to verify results

• Non-specific binding to tissue components:

  • Use appropriate blocking sera matched to secondary antibody host

  • Consider tissue-specific optimization of blocking conditions

• Post-translational modifications:

  • Phosphorylation states may affect epitope recognition

  • Consider phosphatase treatments in control experiments

How do monoclonal and polyclonal PP2B antibodies compare in research applications?

When selecting between monoclonal and polyclonal antibodies, consider your specific experimental requirements and validation needs .

What design of experiments (DOE) approach should I use to optimize PP2B-B1/2 antibody purification protocols?

A systematic DOE approach for antibody purification optimization:

• Define critical parameters:

  • Process steps (e.g., Pre vs. Post Protein A binding)

  • Residence time (exposure duration to chromatographic resin)

  • Buffer conditions (pH, salt concentration)

  • Elution conditions

• Design multifactor experiments:

  • Use factorial or response surface designs rather than one-factor-at-a-time approaches

  • A 27-run experimental design can efficiently explore four factors at 2-3 levels each

  • This approach reduces optimization time from 6+ months to weeks

• Analyze interactions between factors:

  • Statistical analysis enables detection of synergistic or antagonistic effects

  • Software tools like Design-Expert® facilitate optimal experimental design

• Validation studies:

  • Confirm optimized conditions with larger-scale runs

  • Verify antibody functionality in downstream applications

How can I develop a quantitative B-cell ELISpot assay to measure specific immune responses to PP2B epitopes?

To develop a quantitative B-cell ELISpot assay for PP2B epitope-specific responses:

• Optimization steps:

  • Begin by establishing the assay using a hybridoma cell line specific for the epitope

  • Determine optimal coating concentration of capture antibody

  • Optimize cell density and incubation conditions

  • Establish appropriate development reagents and times

• B-cell enumeration approach:

  • Quantify both antibody-secreting cells and memory B cells

  • Use sequential immunizations with heterologous scaffolds to immuno-focus responses

  • Compare homologous vs. heterologous scaffolds to assess specificity

• Validation methods:

  • Confirm epitope-specific antibody responses by ELISA

  • Correlate B-cell frequency with serum antibody levels

  • Analyze quality of antibodies induced by different immunization strategies

This approach has been successfully implemented for analyzing epitope-specific B-cell responses in various immune contexts .

Bibliography

Selection of key references supporting these methodological recommendations. For complete citations, please refer to the numbered sources in the text.

• Santa Cruz Biotechnology. PP2B-B1/2 Antibody (D-1) Technical Information.

• Thrombosis and Haemostasis. Antibodies to factor XII are distinct from antibodies to prothrombin in patients with the anti-phospholipid syndrome.

• R&D Systems. Primary Antibody Selection & Optimization.

• PLAbDab. The Patent and Literature Antibody Database: an evolving reference set of functionally diverse, literature-annotated antibody sequences and structures.

• Flow Cytometry Core. Relevance of Antibody Validation and Titration for Flow Cytometry.

• PMC. Protein Phosphatase PP2C Identification in Entamoeba spp.

• PMC. P12-12. Analysis of antibody and B cell responses following immunization with scaffold proteins displaying the HIV-1 gp41 2F5 epitope.

• Design of Experiments. DOE-Catalyzes-Optimization-of-Purification-Process-for-Monoclonal Antibodies.

• PMC. Antibody tests for identification of current and past infection with SARS‐CoV‐2.

• PMC. Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction.

• PMC. False Positive Class II HLA Antibody Reaction Due to Antibodies Against Denatured Class II Molecules.