DPEP1 Antibody, HRP conjugated

What is DPEP1 and why is it significant for kidney research?

DPEP1 (Dipeptidase 1) is a glycosylphosphatidylinositol-anchored, disulfide-linked, glycosylated homodimer highly expressed in the brush border of proximal tubular epithelial cells. Its significance spans multiple functions:

• Hydrolyzes a wide range of dipeptides and is involved in glutathione and leukotriene metabolism

• Metabolizes the beta-lactam ring of thienamycin antibiotics and is the target of the enzymatic inhibitor cilastatin

• Recently identified as a major leukocyte adhesion receptor in the kidney, lungs, and liver during inflammatory conditions

• Plays a critical role in renal inflammation and acute kidney injury (AKI)

For researchers, DPEP1 represents a promising therapeutic target for preventing AKI, making antibodies against this protein valuable research tools.

What applications are suitable for DPEP1 Antibody, HRP conjugated?

DPEP1 Antibody, HRP conjugated is optimized for several research applications:

Methodological approach: When using for ELISA, remember this antibody works through competitive enzyme immunoassay technique. The assay sample and buffer are incubated with DPEP1-HRP conjugate, where the intensity of the resulting color is inversely proportional to DPEP1 concentration since DPEP1 from samples and DPEP1-HRP conjugate compete for anti-DPEP1 antibody binding sites .

How should samples be prepared for optimal DPEP1 detection?

Sample preparation varies by tissue type and experimental goals:

For serum samples:

• Collect serum and allow samples to clot for 30 minutes at room temperature

• Centrifuge at 1,000 × g for 15 minutes

• Assay immediately or aliquot and store at -20°C or -80°C

For plasma samples:

• Collect using EDTA or heparin as an anticoagulant

• Centrifuge samples at 1,000 × g (3,000 rpm) at 4°C within 30 minutes of collection

• Aliquot and store at -20°C or -80°C

For tissue homogenates:

• Rinse tissues in ice-cold PBS to remove excess blood

• Weigh approximately 300-500 mg of tissue

• Mince to small pieces and homogenize in 500 μL of PBS

• Subject to ultrasonication or freeze-thaw cycles to break cell membranes

• Centrifuge for 15 minutes at 1,500 × g and collect supernatant

How does DPEP1 expression change during kidney injury and what detection considerations apply?

DPEP1 expression undergoes significant changes during kidney injury:

• In normal human kidney, DPEP1 is primarily expressed in the proximal tubule

• During ischemia-reperfusion injury (IRI), DPEP1 expression increases in peritubular capillaries

• DPEP1 protein expression in total kidney homogenates increases within 8 hours of IRI or systemic lipopolysaccharide administration

• IRI and LPS induce a slight increase in DPEP1 molecular mass consistent with additional posttranslational modifications

Methodological considerations:
When studying injury models, researchers should:

• Include time-course analysis (0h, 8h, 24h, 48h post-injury) to capture expression dynamics

• Use both whole kidney lysates and isolated tubular fractions to distinguish compartment-specific changes

• Consider using lower antibody dilutions (1:1000 rather than 1:5000) for early timepoints when expression changes are subtle

• Include appropriate controls (sham surgery or vehicle treatment) to account for procedural effects

How can researchers optimize ELISA protocols using DPEP1 Antibody, HRP conjugated?

The competitive ELISA technique using DPEP1-HRP conjugated antibody requires specific optimization:

Protocol optimization steps:

• Calibration curve preparation: Use serial dilutions of known DPEP1 standards (0-100 ng/mL) to establish the working range

• Sample dilution testing: Test neat (undiluted) samples alongside 1:2 and 1:4 dilutions to ensure readings fall within the calibration curve

• Incubation optimization: The standard protocol recommends 1-hour incubation of samples with DPEP1-HRP conjugate in pre-coated plates

• Washing optimization: Implement 5 complete wash cycles to minimize background

• Substrate reaction timing: Carefully time the substrate-enzyme reaction (typically 15-30 minutes) for optimal signal-to-noise ratio

Quality control metrics:

• Intra-assay CV% should be <5.6%

• Inter-assay CV% should be <7.9%

• Spike recovery should fall between 94-103%

• Dilutional linearity should maintain between 92-108%

• Sensitivity threshold reported at 0.1 ng/mL

What is the role of DPEP1 in leukocyte adhesion and how can it be studied using available antibodies?

Recent research has revealed DPEP1's novel function as a leukocyte adhesion receptor:

• DPEP1 deficiency or inhibition primarily blocks neutrophil adhesion to peritubular capillaries

• It reduces inflammatory monocyte recruitment to the kidney after ischemia-reperfusion injury

• DPEP1 works alongside CD44 and ICAM-1 in recruiting monocyte/macrophages to the kidney following IRI

• Endothelial cell activation is required to modulate DPEP1's leukocyte adhesion capability

Methodological approach for study:

• In vivo studies: Use DPEP1 knockout mice (Dpep1^-/-) or pretreat with DPEP1 antagonists (including LSALT peptide)

• Flow cytometry analysis: Quantify GFP+ leukocyte subpopulations in kidney tissue

• Immunohistochemistry: Use anti-DPEP1 antibodies at 1:5000 dilution followed by HRP-conjugated secondary antibodies to visualize expression in tissue sections

• Comparative adhesion studies: Compare effects of DPEP1 inhibition versus CD44 or ICAM-1 blockade on neutrophil and monocyte recruitment

How can researchers validate DPEP1 Antibody specificity in experimental systems?

Antibody validation is crucial for ensuring experimental rigor:

Multiple validation approaches:

• Western blot validation: Test antibody against recombinant DPEP1 protein alongside positive control samples (HepG2 cells, mouse liver tissue, HEK-293 cells)

• Knockout/knockdown controls: Use DPEP1 knockout or siRNA knockdown samples as negative controls

• Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm specificity

• Cross-reactivity testing: Confirm reactivity with human samples while testing potential cross-reactivity with mouse and rat samples

• Immunohistochemical validation: Confirm localization patterns match known DPEP1 distribution (apical surface of colon epithelium, proximal tubules in kidney)

What considerations apply when using DPEP1 Antibody, HRP conjugated for investigating DPEP1's enzymatic versus adhesion functions?

DPEP1 has dual functions - enzymatic activity and adhesion receptor capacity:

Experimental design considerations:

• Function-specific inhibitors:

  • Use cilastatin to inhibit DPEP1's enzymatic activity without affecting adhesion

  • Use LSALT peptide as a non-enzymatic DPEP1 inhibitor targeting adhesion functions

• Read-out selection:

  • For enzymatic studies: Measure substrate conversion (dipeptide hydrolysis, leukotriene D4 conversion to E4)

  • For adhesion studies: Quantify neutrophil/monocyte adhesion or recruitment

• Molecular weight considerations:

  • Normal DPEP1 appears at approximately 46 kDa

  • Post-translationally modified DPEP1 (associated with adhesion function) shows slightly higher molecular weight

• Experimental models:

  • For enzymatic studies: In vitro enzyme assays with purified protein

  • For adhesion studies: Ex vivo flow chamber assays or in vivo models of inflammation

What common issues might researchers encounter when using DPEP1 Antibody, HRP conjugated and how can they be resolved?

Methodological approach to optimization:
When troubleshooting, systematically test one variable at a time:

• Run positive control samples (HepG2 cells, mouse liver tissue) alongside experimental samples

• Include a calibration curve with each assay run

• Document lot-to-lot variation by maintaining reference samples

• For competitive ELISA, ensure equilibrium between sample DPEP1 and DPEP1-HRP conjugate

How do different storage conditions affect DPEP1 Antibody, HRP conjugated performance?

Proper storage is critical for maintaining antibody activity:

• Store at -20°C or -80°C to avoid activity loss

• Avoid repeated freeze-thaw cycles by preparing single-use aliquots

• Storage buffer composition typically includes:

  • 0.01M PBS, pH 7.4

  • 0.03% Proclin-300 as preservative

  • 50% glycerol for stabilization

• Working solutions can be stored at 4°C for up to one week

• Some formulations include 0.1% BSA for additional stability in small volume (20μL) aliquots

For long-term studies, researchers should validate each new lot against reference standards to ensure consistent performance.

How is DPEP1 Antibody, HRP conjugated used in acute kidney injury research?

DPEP1 antibodies enable critical research into AKI mechanisms:

• Expression analysis: DPEP1 protein expression increases in kidney homogenates within 8 hours of IRI

• Therapeutic targeting studies: DPEP1 antagonists (including LSALT peptide) abrogate ischemia reperfusion-induced AKI

• Mechanistic investigations: DPEP1 inhibition primarily blocks neutrophil adhesion to peritubular capillaries and reduces inflammatory monocyte recruitment

• Combination therapy research: DPEP1 inhibition shows additive effects with ICAM-1 blockade in reducing leukocyte recruitment

Methodological approach:

• Use DPEP1 antibodies to quantify expression changes following injury

• Employ flow cytometry with GFP+ leukocyte markers to quantify cell-specific responses

• Combine with kidney function assessments (blood urea nitrogen, serum creatinine) to correlate molecular changes with functional outcomes

What considerations apply when investigating DPEP1's role in cancer using HRP-conjugated antibodies?

DPEP1 has emerging roles in cancer research:

• Immunohistochemical analysis shows DPEP1 expression in colon cancer tissue

• DPEP1 expression patterns may differ between normal and cancerous tissues

• HRP-conjugated antibodies enable sensitive detection in archived FFPE (formalin-fixed paraffin-embedded) samples

Methodological considerations:

• Tissue processing: Optimize antigen retrieval methods (heat-induced vs. enzymatic)

• Antibody dilution: For IHC applications in cancer tissues, higher dilutions (1:5000) may provide better signal-to-noise ratio

• Counterstaining: Use appropriate nuclear counterstains to visualize tissue architecture

• Controls: Include normal adjacent tissue within the same section as internal control

• Quantification: Employ digital image analysis for objective quantification of expression levels and patterns

How might DPEP1 Antibody, HRP conjugated be utilized in emerging research on DPEP1 as a therapeutic target?

As DPEP1 emerges as a therapeutic target, HRP-conjugated antibodies will play crucial roles:

• Target validation: Confirming DPEP1's role in different disease models beyond AKI (potentially in sepsis and other inflammatory conditions)

• Pharmacodynamic biomarker: Measuring changes in DPEP1 expression or modification during experimental therapeutic interventions

• Mechanism studies: Distinguishing between DPEP1's enzymatic versus adhesion receptor functions

• Therapeutic antibody development: Providing templates for therapeutic antibody design targeting specific DPEP1 epitopes

Methodological approaches:

• Utilize HRP-conjugated antibodies in high-throughput screening of DPEP1 inhibitor candidates

• Develop quantitative ELISA assays for measuring DPEP1 in biological fluids during clinical trials

• Apply in cellular and tissue assays to confirm target engagement of novel therapeutics

What novel applications might be developed for simultaneous detection of DPEP1 with other kidney injury markers?

Multi-marker approaches represent the future of kidney injury research:

• Multiplex assay development: Combining DPEP1 with established kidney injury markers (KIM-1, NGAL, IL-18)

• Spatial profiling: Using antibody panels to map expression patterns across different kidney compartments

• Temporal dynamics: Tracking the sequence of marker expression changes during injury progression and recovery

Methodological considerations:

• Develop protocols that balance optimal conditions for multiple antibodies

• Consider sequential rather than simultaneous detection for HRP-conjugated antibodies

• Explore alternative detection methods (fluorescence) for multiplexing

• Validate marker relationships across different injury models and severity levels