PPDPF Antibody

What types of PPDPF antibodies are available for research purposes?

Based on the available literature, researchers have successfully used polyclonal anti-rabbit PPDPF antibodies in their studies. Specifically, PPDPF antibodies from Proteintech (catalog number 19912-1-AP for lung cancer studies and referenced in HCC research) have been validated in multiple experimental settings including western blotting (1:1000 dilution) and immunohistochemistry (1:100 dilution) . Both studies demonstrate that these antibodies can reliably detect PPDPF expression in tissue samples and cell lines.

What are the recommended applications for PPDPF antibodies?

PPDPF antibodies have been successfully employed in several experimental techniques:

• Western blotting (WB) - For protein expression analysis in cell and tissue lysates at 1:1000 dilution

• Immunohistochemistry (IHC) - For visualization of PPDPF in formalin-fixed, paraffin-embedded tissue sections at 1:100 dilution

• Immunoprecipitation (IP) - For studying protein-protein interactions, particularly when investigating PPDPF binding partners like BABAM2

These applications have been validated in lung cancer cell lines (A549, H1299, H157, H358, and H520), normal bronchial epithelial cells (Beas-2B), and HCC tissue specimens .

What are the ideal storage conditions for PPDPF antibodies?

While specific storage conditions were not explicitly stated in the search results, standard antibody storage practices should be followed. Generally, antibodies should be stored at -20°C for long-term storage and 4°C for short-term use. Aliquoting antibodies to avoid repeated freeze-thaw cycles is recommended to maintain antibody integrity and specificity. When preparing working dilutions, use buffers containing protein stabilizers like BSA and sodium azide as a preservative.

How does PPDPF interact with BABAM2 and MDM2 in radioresistance pathways?

Research has revealed that PPDPF promotes radioresistance in lung cancer cells through a specific molecular mechanism. PPDPF directly interacts with BABAM2, an antiapoptotic protein, as demonstrated through co-immunoprecipitation experiments . This interaction prevents BABAM2 from being ubiquitinated by MDM2, thereby stabilizing BABAM2 and enhancing its antiapoptotic functions .

Mechanistically, when lung cancer cells overexpress PPDPF, they show:

• Decreased apoptosis following radiation treatment

• Reduced expression of cleaved Caspase 3

• Increased expression of Bcl-xl and BABAM2

• Lower levels of γH2AX (a marker of DNA damage) after irradiation

• Faster restoration of γH2AX to basal levels, indicating enhanced DNA repair capacity

These findings suggest that targeting the PPDPF-BABAM2-MDM2 axis could potentially sensitize lung cancer cells to radiotherapy, making it a valuable research direction for improving cancer treatment efficacy.

How should researchers quantify and interpret PPDPF expression in tissue samples?

For quantification of PPDPF expression in tissue samples, researchers have employed several complementary approaches:

• mRNA quantification: Real-time PCR using SYBR Green can be performed with the following primers:

  • Forward: 5′-CGGTCTTCTCTGCAAATGGGC-3′

  • Reverse: 5′-TGGCTGGTGGGATCTGGG-3′

• Protein quantification: Western blotting using anti-PPDPF antibody (Proteintech, 19912-1-AP, 1:1000)

• Tissue expression patterns: Immunohistochemistry with anti-PPDPF antibody (1:100 dilution) evaluated using the German semiquantitative scoring system

For interpretation, researchers should consider that:

What are the considerations for using PPDPF antibodies in co-immunoprecipitation experiments?

When designing co-immunoprecipitation (Co-IP) experiments to study PPDPF interactions, researchers should consider:

• Antibody selection: For detecting interactions between PPDPF and binding partners (e.g., BABAM2), researchers have successfully used anti-MYC antibody (Proteintech, 16286-1-AP, 1:2000) for MYC-tagged PPDPF and anti-Flag antibody (Sigma, F1804) for Flag-tagged BABAM2 .

• Lysis conditions: Use IP lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% NP-40, and protease/phosphatase inhibitors to preserve protein-protein interactions .

• Incubation parameters: After collecting the supernatant, incubate with antibody-coupled beads for 4 hours, followed by three washes with wash buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% NP-40) .

• Controls: Include appropriate negative controls (IgG or empty vector) and input samples to validate specific interactions.

• Detection: For western blotting after Co-IP, use anti-PPDPF (1:1000) and anti-BABAM2 (1:1000) antibodies to confirm the interaction .

How can PPDPF antibodies be used to evaluate radioresistance in cancer cells?

PPDPF antibodies can be instrumental in studying radioresistance mechanisms through several experimental approaches:

• Expression analysis: Compare PPDPF levels before and after radiation treatment using western blotting (1:1000 dilution) .

• Apoptosis assessment: Evaluate how PPDPF expression affects radiation-induced apoptosis by measuring:

  • Cleaved Caspase 3 levels

  • Bcl-xl expression

  • BABAM2 expression

• DNA damage repair evaluation: Monitor γH2AX expression kinetics (a marker of DNA double-strand breaks) after radiation in cells with different PPDPF expression levels. In cells overexpressing PPDPF, γH2AX levels decrease more rapidly, indicating enhanced DNA repair capacity .

• Colony formation assay: Assess radiation sensitivity by determining survival rates of cancer cells with modified PPDPF expression after different radiation doses .

• Mechanistic studies: Use PPDPF antibodies in Co-IP experiments to identify radiation-induced changes in PPDPF interactions with partners like BABAM2 .

These approaches can provide comprehensive insights into how PPDPF contributes to radioresistance and may help identify strategies to improve radiotherapy outcomes.

What protocols are recommended for immunohistochemical detection of PPDPF in tissue samples?

For optimal immunohistochemical detection of PPDPF in tissue samples, the following protocol is recommended based on successful previous studies:

• Sample preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections (4 μm thickness)

  • Deparaffinize and rehydrate sections using standard procedures

  • Perform antigen retrieval (specific conditions not mentioned in the search results, but typically involves heat-induced epitope retrieval in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Primary antibody incubation:

  • Use anti-PPDPF monoclonal antibody (Proteintech) at 1:100 dilution

  • Incubate overnight at 4°C

• Detection system:

  • Employ Envision Plus Detection Kit (DAKO) following the manufacturer's protocol

  • Counterstain nuclei with hematoxylin

• Controls:

  • Include negative controls by omitting the primary antibody

  • Consider using tissues known to express PPDPF as positive controls

• Evaluation:

  • Use the German semiquantitative scoring system, which considers both staining intensity and area extent

  • Have multiple independent pathologists evaluate the staining to ensure reliability and reduce bias

What is the recommended protocol for detecting PPDPF in cell lysates via western blotting?

For western blot detection of PPDPF in cell lysates, the following protocol has been successfully implemented:

• Sample preparation:

  • Lyse cells using appropriate lysis buffer (specific buffer not detailed in search results, but standard RIPA buffer with protease inhibitors is commonly used)

  • Determine protein concentration using a protein assay method

• SDS-PAGE and transfer:

  • Load equal amounts of protein per lane (typically 20-50 μg)

  • Separate proteins using SDS-PAGE

  • Transfer to nitrocellulose membranes (Bio-Rad)

• Antibody incubation:

  • Block membranes with appropriate blocking buffer

  • Incubate with anti-PPDPF antibody (Proteintech, 19912-1-AP) at 1:1000 dilution

  • Wash membranes and incubate with HRP-conjugated secondary antibodies (Millipore)

• Detection:

  • Visualize using ECL kit (Pierce)

  • Analyze using one-dimensional image analysis software

• Controls:

  • Include loading controls such as anti-HSP90 (Santa Cruz, sc-69703, 1:3000), anti-tubulin (Santa Cruz, sc-5286, 1:4000), or anti-GAPDH (Santa Cruz, sc-47724, 1:4000)

  • Consider including positive controls (cells known to express PPDPF) and negative controls (cells with PPDPF knockdown)

What are common challenges when using PPDPF antibodies and how can they be addressed?

While specific troubleshooting information for PPDPF antibodies was not provided in the search results, researchers may encounter several common challenges when working with antibodies for relatively less-studied proteins:

• Low signal intensity in western blots:

  • Increase antibody concentration (start with 1:500 instead of 1:1000)

  • Optimize incubation time and temperature (e.g., overnight at 4°C instead of 1-2 hours at room temperature)

  • Use enhanced chemiluminescence detection systems with higher sensitivity

  • Increase protein loading amount

• High background in immunohistochemistry:

  • Optimize blocking conditions (try different blocking agents like BSA, normal serum, or commercial blocking solutions)

  • Reduce primary antibody concentration (try 1:200 instead of 1:100)

  • Increase washing duration and frequency

  • Use more specific detection systems

• Inconsistent results across experiments:

  • Standardize lysate preparation methods

  • Use the same antibody lot when possible

  • Implement rigorous positive and negative controls

  • Consider using recombinant PPDPF protein as a standard

• Co-IP efficiency issues:

  • Optimize lysis conditions to preserve protein interactions

  • Try crosslinking before lysis

  • Adjust salt and detergent concentrations in wash buffers

  • Consider tagged versions of PPDPF for enhanced pulldown efficiency

How can researchers validate PPDPF antibody specificity?

Ensuring antibody specificity is crucial for generating reliable research data. For PPDPF antibodies, validation can be performed through several approaches:

• Genetic validation:

  • Compare antibody signal between wild-type cells and cells with PPDPF knockdown using shRNA (as demonstrated in the lung cancer study)

  • Use cells overexpressing PPDPF as positive controls

• Peptide competition assay:

  • Pre-incubate the antibody with excess PPDPF peptide before application

  • A specific antibody will show diminished signal after peptide blocking

• Multi-technique confirmation:

  • Verify that PPDPF detection across different techniques (western blot, IHC, IF) shows consistent patterns

  • Check correlation between mRNA levels (by RT-PCR) and protein levels (by western blot)

• Cross-validation with different antibodies:

  • If available, compare results using antibodies from different suppliers or those targeting different epitopes of PPDPF

• Mass spectrometry confirmation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the precipitated protein

How might PPDPF antibodies be used in developing therapeutic strategies for cancer?

PPDPF antibodies could play crucial roles in developing novel therapeutic strategies for cancer, particularly those targeting radioresistance mechanisms:

• Target validation:

  • Use PPDPF antibodies to confirm the presence and expression levels of PPDPF in patient-derived xenografts and primary tumor samples

  • Correlate PPDPF expression with treatment response to identify patients who might benefit from PPDPF-targeted therapies

• Mechanism exploration:

  • Employ PPDPF antibodies to elucidate further downstream effects of PPDPF inhibition

  • Investigate how PPDPF interacts with other proteins involved in radioresistance beyond BABAM2

• Therapeutic development:

  • Use antibodies to screen for small molecule inhibitors that disrupt PPDPF-BABAM2 interaction

  • Evaluate the combination of MDM2 inhibitors with radiotherapy in contexts of high PPDPF expression

• Response monitoring:

  • Develop PPDPF antibody-based assays to monitor treatment response in clinical trials

  • Use immunohistochemistry with PPDPF antibodies to stratify patients for personalized treatment approaches

• Diagnostic applications:

  • Explore the potential of PPDPF as a diagnostic or prognostic biomarker for HCC and NSCLC, leveraging antibody-based detection methods

The research indicates that PPDPF may be a valuable therapeutic target, particularly for overcoming resistance to radiotherapy in lung cancer patients .

What experimental models are most appropriate for studying PPDPF function using antibody-based techniques?

Based on the available research, several experimental models have proven effective for studying PPDPF function:

• Cell line models:

  • Lung cancer cell lines: A549, H1299, H157, H358, and H520 have been successfully used to study PPDPF's role in radioresistance

  • Normal bronchial epithelial cell line: Beas-2B provides an important non-cancerous control

  • HCC cell lines (though specific lines were not mentioned in the search results)

• Animal models:

  • The KrasG12D; LKB1f/f mouse model of lung cancer has been employed to study PPDPF function in vivo

  • Consider patient-derived xenograft models to better recapitulate the tumor microenvironment

• Clinical samples:

  • Tissue microarrays containing multiple patient samples allow for high-throughput analysis of PPDPF expression

  • The lung cancer tissue array containing 90 lung cancer tissues and 88 paired adjacent tissues has been effectively used for PPDPF studies

  • HCC tissue specimens and adjacent normal liver tissues from surgical resections provide valuable clinical correlation

• Genetic manipulation models:

  • Stable cell lines with PPDPF overexpression (using Flag-tagged PPDPF)

  • PPDPF knockdown models using shRNA with target sequences:

    • shPPDPF #1: 5'-CCGGTCCTGACCTGAGCGGTTACCACTCGAGTGGTAACCGCTCAGGTCAGGATTTTT-3'

    • shPPDPF #3: 5'-CCGGGGGTTCCACTTCCAGCAACACTCGAGTGTTGCTGGAAGTGGAACCCATTTTT-3'

These models, combined with appropriate antibody-based techniques (western blot, IHC, Co-IP), provide comprehensive platforms for investigating PPDPF's role in cancer progression and therapy resistance.