Phospho-PTPRC (Ser1007) Antibody

Basic Research Questions

• What is PTPRC/CD45 and why is its phosphorylation important in immunological research?

PTPRC (Protein Tyrosine Phosphatase Receptor Type C), commonly known as CD45 or leukocyte common antigen (LCA), is a transmembrane glycoprotein expressed on nearly all nucleated hematopoietic cells, comprising approximately 10% of all surface proteins in lymphocytes. CD45 functions as an essential regulator of T and B cell antigen receptor-mediated activation and plays a critical role in immune cell signaling pathways .

The phosphorylation state of CD45, particularly at serine 1007 (sometimes referenced as serine 1009 in current literature), represents an important post-translational modification that affects CD45 function. Phospho-specific antibodies that detect CD45 only when phosphorylated at Ser1007 are valuable tools for studying how this modification influences immune cell signaling .

• What applications can Phospho-PTPRC (Ser1007) Antibody be used for in immunological research?

Phospho-PTPRC (Ser1007) Antibody can be utilized across multiple experimental applications:

Researchers should note that optimal dilutions may vary by application and specific experimental conditions, and validation in your specific system is recommended .

• What species reactivity does Phospho-PTPRC (Ser1007) Antibody exhibit?

Based on multiple product datasheets, Phospho-PTPRC (Ser1007) Antibody demonstrates reactivity with:

• Human (primary validated species)

• Mouse

• Rat

Some products also show predicted reactivity with:

• Cow

• Pig

• Dog

• Rabbit

It's important to note that sensitivity may vary between species, with some antibodies showing decreased sensitivity in mouse and rat samples compared to human samples .

Intermediate Research Questions

• How does TNF treatment affect CD45 phosphorylation at Ser1007 and what experimental controls should be included?

TNF (Tumor Necrosis Factor) treatment has been shown to induce phosphorylation of CD45 at Ser1007. Western blot analysis of extracts from HeLa cells treated with TNF consistently shows increased phosphorylation at this site .

Recommended experimental controls:

• Negative control: Untreated cells to establish baseline phosphorylation

• Specificity control: Treatment of blots with the phospho-specific peptide used for antibody generation, which should abolish the signal (as demonstrated in validation images where the right lane shows signal elimination after peptide competition)

• Loading control: Detection of total CD45 using a phosphorylation-independent antibody

• Time course: Treatment with TNF at 20ng/ml for varying time points (commonly 15 minutes has been validated)

This experimental approach allows researchers to specifically assess the impact of TNF-induced signaling on CD45 phosphorylation status.

• What are the optimal experimental conditions for using Phospho-PTPRC (Ser1007) Antibody in Western blotting?

For optimal Western blotting results with Phospho-PTPRC (Ser1007) Antibody:

Sample preparation:

• Use freshly prepared cell lysates from TNF-treated cells (20ng/ml, 15 minutes) as positive control

• Include phosphatase inhibitors in lysis buffer to preserve phosphorylation status

• Recommended cell lines: HeLa cells, Jurkat cells, human spleen tissue

Technical parameters:

• Antibody dilution: 1:500-1:1000 (optimal range based on multiple product datasheets)

• Blocking: 5% BSA in TBST (preferred over milk for phospho-specific antibodies)

• Incubation: Overnight at 4°C for primary antibody

• Expected molecular weight: 147-150 kDa

• Validation control: Include antigen-specific peptide competition

Troubleshooting low signal:

• Increase antibody concentration

• Extend primary antibody incubation time

• Enhance signal with high-sensitivity detection reagents

• Concentrate protein sample through immunoprecipitation

• How can researchers validate the specificity of Phospho-PTPRC (Ser1007) Antibody in their experimental system?

Validating antibody specificity is crucial for reliable research results. For Phospho-PTPRC (Ser1007) Antibody, consider these approaches:

• Peptide competition assay: Preincubate antibody with the phosphorylated peptide used as immunogen (sequence around phosphorylation site of serine 1007: D-D-S(p)-D-S). This should abolish specific binding, as demonstrated in validation images .

• Phosphatase treatment control: Treat duplicate samples with lambda phosphatase before immunoblotting to remove phosphorylation. The signal should disappear in phosphatase-treated samples.

• Genetic validation: Use CRISPR/Cas9-edited cells lacking PTPRC expression as a negative control. The technique for generating PTPRC knockout cells has been described in research using podocytes .

• Stimulus-responsive phosphorylation: Compare samples with and without TNF treatment, which has been shown to increase Ser1007 phosphorylation .

• Multiple detection methods: Confirm findings using at least two independent techniques (e.g., Western blot and immunofluorescence).

Advanced Research Questions

• How does phosphorylation at Ser1007 affect CD45 function in immune cell signaling pathways?

CD45 functions as a receptor-type protein tyrosine phosphatase that regulates immune cell signaling, particularly through modulation of Src family kinases (SFKs). Phosphorylation at Ser1007 represents a key regulatory mechanism that affects CD45's protein tyrosine phosphatase (PTP) activity .

When CD45 is phosphorylated at Ser1007, it can alter:

• Regulation of SFK activity: CD45 can dephosphorylate both the kinase-domain phosphotyrosine (Y394 in p56LCK) and the C-terminal phosphotyrosine (Y505 in p56LCK), with opposing effects on kinase activity .

• T cell receptor signaling: Phosphorylation status affects CD45's ability to modulate TCR signal transduction, potentially through changes in its association with membrane microdomains.

• Cross-talk with other signaling pathways: Recent research suggests that TNF-induced phosphorylation at Ser1007 may represent an intersection between inflammatory and immune signaling cascades .

• Immune checkpoint interaction: PTPRC expression correlates with immune checkpoint molecules including PDCD1, PD-L1/CD274, CTLA4, LAG3, and HAVCR2, suggesting its phosphorylation status may influence immunotherapy responses .

Understanding the functional consequences of Ser1007 phosphorylation remains an active area of research, with implications for both basic immunology and therapeutic development.

• What are the technical considerations when using Phospho-PTPRC (Ser1007) Antibody in phosphoproteomic studies?

When incorporating Phospho-PTPRC (Ser1007) Antibody into phosphoproteomic studies, researchers should consider:

Sample preparation considerations:

• Use rapid lysis methods to preserve phosphorylation status

• Include comprehensive phosphatase inhibitor cocktails

• Perform cell stimulation (e.g., TNF treatment) immediately before lysis

• Consider subcellular fractionation to enrich membrane proteins

Antibody-based enrichment:

• Pre-clear lysates to reduce non-specific binding

• Validate antibody specificity using phosphopeptide competition

• Consider sequential immunoprecipitation approaches

• Use site-specific rather than pan-phosphorylation antibodies

Mass spectrometry integration:

• Employ targeted MS approaches for enhanced sensitivity

• Use SILAC or TMT labeling for quantitative comparisons

• Include identification of surrounding phosphopeptides to assess multi-site phosphorylation

• Compare results with phospho-null mutants where possible

Bioinformatic analysis:

• Map phosphorylation sites to functional domains

• Analyze kinase recognition motifs (Ser1007 context: D-D-S-D-S)

• Integrate with interaction network data

• Consider evolutionary conservation of phosphorylation sites

These techniques have been applied in studies examining phosphoproteomes in human kidney podocytes and other cell types .

• How can Phospho-PTPRC (Ser1007) Antibody be used to study the role of CD45 in autoimmune and inflammatory conditions?

CD45 plays critical roles in autoimmune and inflammatory conditions through its regulation of lymphocyte activation thresholds. The Phospho-PTPRC (Ser1007) Antibody provides a valuable tool for studying these contexts:

Research applications in autoimmunity:

• Signaling dysregulation: Compare phosphorylation patterns between healthy controls and patient samples to identify altered regulation

• Therapeutic target identification: Assess how existing or experimental therapeutics affect CD45 phosphorylation status

• Biomarker development: Evaluate whether Ser1007 phosphorylation correlates with disease activity or treatment response

Inflammatory bone disorders:
Research has established connections between CD45 and inflammatory bone diseases. CD45 promotes severe inflammatory bone destruction in mouse models, with pro-IL-1β generation being a key mechanism . These findings suggest potential applications for studying:

• Rheumatoid arthritis pathogenesis

• Inflammatory osteolysis

• Therapeutic interventions targeting CD45 function

Cancer immunotherapy:
PTPRC expression correlates positively with response to immunotherapy, with higher expression in responders compared to non-responders. Patients with high PTPRC expression showed a higher probability (30% vs. 13%) of responding to immune checkpoint therapy . This suggests using Phospho-PTPRC (Ser1007) Antibody to:

• Predict immunotherapy response

• Monitor treatment effects (PTPRC expression increases significantly post-therapy)

• Develop combination strategies targeting CD45 signaling

• What is the relationship between CD45 and its associated protein PTPRCAP, and how can researchers study this interaction?

CD45 (PTPRC) interacts with an associated protein called PTPRCAP (CD45-AP, LPAP), forming a functional complex that regulates immune cell signaling. Researchers investigating this interaction can utilize several complementary approaches:

Experimental strategies:

• Co-immunoprecipitation studies: Use antibodies against PTPRC and PTPRCAP to pull down protein complexes and analyze interaction partners. Available antibodies include polyclonal PTPRCAP antibody (19715-1-AP) validated for WB, IHC, and IF/ICC applications .

• Proximity ligation assays: Visualize and quantify PTPRC-PTPRCAP interactions in situ using antibodies against both proteins.

• Domain mapping: Generate truncation mutants to identify specific regions required for interaction.

• Phosphorylation effects: Use Phospho-PTPRC (Ser1007) Antibody to determine whether phosphorylation status affects PTPRCAP binding.

• Functional assays: Compare signaling outcomes in cells expressing wild-type PTPRC versus phospho-null (S1007A) or phosphomimetic (S1007D) mutants.

Technical considerations:

• PTPRCAP has an observed molecular weight of 28-30 kDa (calculated: 21 kDa)

• Use Jurkat cells, human spleen tissue, or human tonsillitis tissue as positive controls for PTPRCAP expression

• For co-localization studies, validate antibodies carefully to ensure specificity

• Consider the impact of membrane microdomain organization on interaction dynamics