P4H10 Antibody

What is the P4H10 antibody and what epitope does it recognize?

P4H10 is a mouse monoclonal antibody (IgG1 isotype) that specifically recognizes human integrin beta-1 (CD29). The antibody binds to a specific epitope mapped to the amino acid sequence NKGEVFNELVGK (a.a. 207-218) of human integrin beta-1. This antibody was developed using human fibrosarcoma HT-1080 cells as the immunogen and was deposited to the Developmental Studies Hybridoma Bank (DSHB) by researchers from the Fred Hutchinson Cancer Research Center .

What sample preparation methods are compatible with P4H10 for immunostaining?

According to depositor notes, P4H10 antibody performs well with several fixation methods for immunostaining, including:

It is worth noting that the epitope recognized by P4H10 is not trypsin-sensitive, which can be advantageous for certain applications where proteolytic treatment is part of the sample preparation protocol .

What research applications is P4H10 antibody validated for?

P4H10 has been validated for multiple research applications, making it versatile for studying integrin beta-1 biology:

This antibody recognizes all known isoforms of integrin beta-1, enhancing its utility across different experimental systems .

How can P4H10 be implemented in functional studies of cell adhesion?

P4H10 is particularly valuable for functional studies as it exhibits function-blocking activity. According to depositor notes, P4H10 inhibits endodermal cell and keratinocyte attachment to extracellular matrix components including collagen type 1, fibronectin, and laminin. Additionally, it inhibits cell-cell adhesion . This makes it an excellent tool for:

• Investigating integrin β1-dependent cell adhesion to specific ECM components

• Studying the role of integrin β1 in cellular migration

• Examining cell-cell interaction mechanisms

• Analyzing integrin-dependent signaling pathways

A typical functional blocking protocol involves pre-incubating cells with P4H10 (10-20 μg/ml) for 30-60 minutes prior to adhesion assays.

How should researchers address the conflicting reports regarding P4H10 in Western blot applications?

To reconcile these conflicting reports, researchers should consider:

• Performing side-by-side comparisons with other validated anti-integrin β1 antibodies

• Testing different sample preparation methods, particularly using:

  • Non-denaturing conditions

  • Mild detergent extraction

  • Native PAGE rather than SDS-PAGE

• Optimizing blocking conditions (5% BSA often performs better than milk for phosphoproteins)

• Implementing positive controls from cell types known to express high levels of integrin β1

A systematic evaluation of these parameters should be documented before concluding about the antibody's utility for Western blot in your specific experimental system.

What are the technical considerations for using P4H10 in flow cytometry?

When using P4H10 for flow cytometry applications, several technical aspects should be considered:

• Cell preparation: Since P4H10 recognizes native protein conformations, avoid harsh fixation methods that might denature the epitope.

• Titration: Perform antibody titration (typically 0.1-10 μg/ml) to determine optimal concentration for your specific cell type.

• Controls:

  • Include isotype control (mouse IgG1)

  • Use cell lines with known high (e.g., HT-1080) and low/negative expression of integrin β1

  • Consider a blocking experiment to confirm specificity

• Staining protocol example:

  • Harvest cells using enzyme-free dissociation buffer to preserve surface integrins

  • Resuspend 1×10^6 cells in 100 μl staining buffer (PBS + 1% BSA)

  • Add P4H10 (starting at 1 μg per 10^6 cells)

  • Incubate for 30 minutes at 4°C

  • Wash twice with staining buffer

  • Add appropriate fluorophore-conjugated secondary antibody

  • Incubate for 30 minutes at 4°C in the dark

  • Wash twice and analyze

What approaches can be used to validate P4H10 specificity in new experimental systems?

Validating antibody specificity is crucial, particularly when applying P4H10 to new cell types or experimental systems:

• Genetic validation:

  • Use ITGB1 knockout cell lines (e.g., the A-431 knockout line mentioned in comparable antibody validation studies)

  • Employ siRNA or shRNA knockdown of ITGB1

  • Overexpress ITGB1 in low-expressing cell lines

• Peptide competition:

  • Pre-incubate P4H10 with excess synthetic peptide containing the epitope sequence (NKGEVFNELVGK)

  • This should abolish specific binding

• Multiple antibody approach:

  • Compare staining patterns with other validated anti-ITGB1 antibodies targeting different epitopes

  • Concordant results increase confidence in specificity

• Mass spectrometry validation:

  • Perform immunoprecipitation with P4H10 followed by mass spectrometry

  • Confirm ITGB1 as the predominant precipitated protein

How does P4H10 compare to other integrin β1 antibodies in research applications?

When selecting an antibody for integrin β1 research, it's important to consider how P4H10 compares to alternatives:

This comparative information can guide researchers in selecting the most appropriate antibody based on their specific experimental needs and model systems.

What methodological considerations are important when using P4H10 in immunohistochemistry of tissue specimens?

For optimal immunohistochemical staining with P4H10:

• Antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) is recommended

  • Pressure cooker methods may enhance epitope accessibility

• Section thickness:

  • 4-5 μm sections are optimal for most applications

  • Thicker sections may require adjusted incubation times

• Blocking:

  • Include both protein block (e.g., 5% normal goat serum) and endogenous peroxidase block

  • Consider biotin/avidin blocking if using biotin-based detection systems

• Primary antibody incubation:

  • Start with 1:100 dilution (adjust based on titration)

  • Overnight incubation at 4°C often yields best signal-to-noise ratio

• Counterstaining:

  • Light hematoxylin counterstain prevents masking of DAB signal

  • For fluorescence, DAPI nuclear stain provides good contrast

How can P4H10 be used in multiplexed imaging applications?

When incorporating P4H10 into multiplexed immunofluorescence or immunohistochemistry:

• Antibody panel design:

  • Since P4H10 is a mouse IgG1, avoid other mouse IgG1 antibodies in the same panel

  • Consider using antibodies from different host species or different mouse IgG subclasses

• Sequential staining approaches:

  • For same-species antibodies, consider tyramide signal amplification with heat-mediated stripping between rounds

  • Alternatively, directly conjugated P4H10 can avoid secondary antibody cross-reactivity

• Spectral considerations:

  • When selecting fluorophores, account for the cellular distribution of integrin β1 (primarily membrane)

  • Choose spectrally distinct fluorophores for markers expected to co-localize with integrin β1

• Controls for multiplexed imaging:

  • Single-stained controls are essential for spectral unmixing

  • Isotype controls should be included for each antibody class used

What are common issues encountered with P4H10 and their solutions?

How can researchers optimize co-immunoprecipitation protocols using P4H10?

For successful co-immunoprecipitation of integrin β1 complexes using P4H10:

• Lysis conditions:

  • Use mild, non-denaturing lysis buffers (e.g., 1% NP-40 or CHAPS)

  • Include protease and phosphatase inhibitors

  • Maintain physiological pH (7.4-7.6)

• Pre-clearing:

  • Pre-clear lysates with protein G beads to reduce non-specific binding

  • Optional: pre-incubate with irrelevant mouse IgG1

• Antibody binding:

  • Use 2-5 μg of P4H10 per 500 μg of total protein

  • Incubate overnight at 4°C with gentle rotation

• Bead selection:

  • Protein G Sepharose has higher affinity for mouse IgG1 than Protein A

  • Pre-block beads with BSA to reduce non-specific binding

• Washing stringency:

  • Balance between preserving interactions (mild washing) and reducing background (stringent washing)

  • Typically 4-5 washes with decreasing detergent concentration

This approach will help maintain native protein-protein interactions while minimizing non-specific binding.

How is P4H10 being utilized in integrin-targeted therapeutic research?

Recent advances in integrin biology have expanded the applications of function-blocking antibodies like P4H10:

• Cancer research applications:

  • Studying integrin β1's role in cancer cell invasion and metastasis

  • Investigating resistance mechanisms to targeted therapies

  • Exploring combination approaches with integrin inhibition

• Stem cell research:

  • Modulating integrin-ECM interactions during differentiation

  • Investigating niche interactions in stem cell maintenance

  • Optimizing culture conditions for stem cell expansion

• Drug discovery applications:

  • As a tool for validating small molecule integrin β1 inhibitors

  • In phage display studies for developing new therapeutic antibodies

  • For identifying novel integrin β1 interaction partners as drug targets

What considerations are important when using P4H10 in conjunction with light chain shuffling techniques?

The search results reference light chain shuffling for antibody optimization. When considering similar approaches with P4H10 or related antibodies:

• Library construction considerations:

  • Maintain the heavy chain CDRs that primarily determine epitope specificity

  • Design appropriate frameworks for the shuffled light chains

  • Consider the diversity of the light chain library (naïve vs. immune)

• Selection strategy:

  • Define clear parameters for improved antibodies (affinity, specificity, function)

  • Design appropriate screening assays that reflect the intended application

  • Include competitive elution steps to select higher-affinity variants

• Validation requirements:

  • Confirm retained epitope specificity after light chain shuffling

  • Verify improved properties using quantitative assays

  • Test cross-reactivity with related integrins to ensure specificity is maintained

This approach can potentially generate improved variants of P4H10 with enhanced properties for specific research applications .

How can researchers differentiate between P4H10 antibody and P4H10 enzymes in scientific literature?

There is potential confusion in the literature between:

• P4H10 antibody (against integrin β1/CD29)

• P4H10 subfamily of prolyl-4-hydroxylases (enzymes)

To properly differentiate:

• Literature search strategies:

  • Use precise search terms: "P4H10 antibody AND integrin" vs. "P4H10 prolyl hydroxylase"

  • Include catalog numbers or clone identifiers when searching for antibody information

  • Check for contextual clues about whether the reference is to the enzyme or antibody

• Referencing in publications:

  • Clearly specify "P4H10 anti-integrin β1 antibody" rather than just "P4H10"

  • Include clone information, manufacturer, and catalog numbers

  • For the enzyme, use full nomenclature "prolyl-4-hydroxylase P4H10 subfamily"

This distinction is particularly important as recent research has focused on P4H10 enzyme subfamily in plants and their role in O-glycosylation .