RBP7 Antibody

What is the optimal dilution range for RBP7 antibodies in different research applications?

RBP7 antibodies require specific dilutions for optimal results depending on the application. Based on validated protocols, the following dilution ranges are recommended:

These ranges should be considered starting points, as optimal dilutions may vary based on antibody source, tissue type, and detection method. It is recommended that researchers titrate the antibody in each testing system to obtain optimal results .

How can I validate the specificity of an RBP7 antibody for my experiments?

Validating antibody specificity is crucial for reliable results. For RBP7 antibodies, consider these methodological approaches:

• Positive control tissues/cells: Use tissues with known RBP7 expression such as HEK-293 cells, mouse adipose tissue, human kidney tissue, or human colon cancer tissue .

• Multiple detection methods: Validate using different methods (WB, IHC, IF) to confirm consistent target recognition.

• Knockdown validation: Compare staining between wild-type samples and those with RBP7 knockdown (using siRBP7). Researchers have successfully verified RBP7 antibody specificity by examining protein expression changes after siRNA knockdown in 3T3-L1 cells .

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight (15.5 kDa calculated, 15-18 kDa observed) .

• Cross-reactivity testing: Verify reactivity with your species of interest, as some RBP7 antibodies show reactivity with human and mouse samples, while others are species-specific .

What are the recommended storage conditions for RBP7 antibodies?

For maximum stability and activity retention of RBP7 antibodies:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• Most commercial preparations are stable for one year after shipment when properly stored

• For some formulations, aliquoting is unnecessary for -20°C storage

• Most RBP7 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations (20μl sizes) may contain 0.1% BSA

Proper storage conditions are essential as repeated freeze-thaw cycles can diminish antibody activity and specificity over time.

Which tissues demonstrate significant RBP7 expression, and how can this guide experimental design?

RBP7 exhibits a distinctive tissue-specific expression pattern that researchers should consider when designing experiments:

• Endothelium-specific expression: RBP7 is essentially endothelium-specific or virtually so, particularly enriched in small blood vessels . This makes RBP7-deficient mice an effective endothelial cell-specific RBP7-deficient model.

• Adipose tissue expression: RBP7 is abundantly expressed in both white and brown mouse adipose tissues, with higher expression in adipocytes compared to stromal vascular cells .

• Regional tumor differences: In colon cancer, tumor cells at the tumor-stroma interface (tumor edge) show significantly higher RBP7 expression compared to cells in the tumor center . This spatial heterogeneity is important when analyzing tumor samples.

When designing experiments:

• Include both edge and center regions when analyzing tumor tissues

• Use MACS (magnetic-activated cell sorting) to reliably isolate RBP7-expressing endothelial cells (validated by co-expression of CD31 and NOS3)

• Consider tissue-specific controls based on known expression patterns

How can I quantitatively assess RBP7 expression in tumor tissue specimens?

For robust quantification of RBP7 expression in tumor samples:

• H-score calculation: Use automated detection algorithms (like QuPath) to calculate H-scores based on extent and intensity of RBP7 nuclear staining. The formula incorporates: 3× % of strongly stained tumor cells + 2× % of moderately stained tumor cells + 1× % of weakly stained tumor cells .

• Regional assessment: Quantify RBP7 expression separately in tumor cells at the tumor-stroma interface (tumor edge) and 100 μm or more away from the interface (tumor center) .

• Computational color deconvolution: Apply this technique to separate haematoxylin and DAB stains for more accurate quantification .

• Blinded analysis: Conduct all analyses blinded to clinical outcome to prevent bias .

In colon cancer specimens, this approach revealed H-scores ranging from 0 to 184.27, demonstrating wide variation in RBP7 expression among different tumors .

How can I effectively study RBP7's role in cardiovascular stress response using animal models?

RBP7 plays a protective role against oxidative stress in response to cardiovascular stressors. To study this:

• Dietary challenge model: Feed RBP7-deficient and control mice with high-fat diet (HFD) for 10-12 weeks. RBP7-deficient mice exhibit severe endothelial dysfunction in response to HFD without differences in weight gain, glucose homeostasis, or hepatosteatosis .

• Angiotensin II model: Administer subpressor doses of Ang-II (140 ng/kg/min) to RBP7-deficient and control mice for 2 weeks. This approach reveals RBP7's protective role without causing significant blood pressure differences between groups .

• Functional vascular assessment: Measure endothelium-dependent relaxations in response to acetylcholine in isolated aortic rings. RBP7-deficient mice show impaired relaxation response during cardiovascular stress .

• Oxidative stress mechanism: Include experiments with the superoxide scavenger tempol (1 mmol/L) to determine if oxidative stress mediates observed dysfunction. Tempol can ameliorate impairment in acetylcholine-induced relaxation in RBP7-deficient vessels .

• Blood pressure monitoring: Use both tail cuff and radiotelemetry methods for comprehensive assessment. No significant difference in blood pressure should be observed between control and RBP7-deficient mice at baseline .

What experimental approaches can be used to investigate RBP7's role in adipogenesis?

To study RBP7's function in adipocyte differentiation:

• Gene modulation in 3T3-L1 cells:

  • Overexpress mouse Rbp7 using lentiviral vectors (verified by qPCR and Western blot)

  • Knockdown Rbp7 using siRNA transfection

  • Induce adipogenic differentiation 2 days after transfection

• Adipocyte differentiation assessment:

  • Morphological evaluation of lipid accumulation

  • Triglyceride content measurement (significantly higher in RBP7-overexpressing cells compared to controls)

  • Expression analysis of adipogenic markers (PPARγ, FABP4)

• Retinoid metabolism analysis:

  • RARE luciferase reporter assay to measure RA signaling activity

  • Expression analysis of retinoid metabolism genes:

    • Retinol storage: Lrat

    • RA synthesis: Raldh1

    • RA degradation: Cyp26a1

• Protein expression verification: Western blot analysis of LRAT, ALDH1A1, and CYP26A1 proteins using specific antibodies

Research has shown that RBP7 overexpression significantly increases RARE-Luc reporter activity and expression of genes involved in retinol metabolism, while RBP7-deficient adipocytes show opposite effects .

How can RBP7 antibodies be utilized to investigate its role in breast cancer progression?

RBP7 exhibits significant roles in breast cancer, particularly in ER-positive subtypes. To investigate:

• Expression analysis in clinical samples:

  • Compare RBP7 protein and mRNA expression between normal breast tissue and breast cancer samples

  • Stratify analysis by ER status (ER+ vs. ER-) as RBP7 has prognostic value particularly in ER+ breast cancer patients

• Promoter methylation analysis:

  • Investigate correlation between RBP7 expression and its promoter methylation status

  • Low RBP7 expression correlates with promoter hypermethylation in breast cancer

• Pathway investigation:

  • Focus on PPAR pathway and PI3K/AKT pathway components

  • Functional enrichment analysis has shown that downregulation of RBP7 expression influences breast cancer through these pathways

• Survival correlation:

  • Correlate RBP7 expression levels with patient prognosis

  • Lower expression of RBP7 correlates with worse prognosis in ER+ breast cancer patients

• Antibody validation for breast tissue:

  • Optimize antibody concentrations specifically for breast tissue specimens

  • Include appropriate positive and negative controls based on known expression patterns

What methodological considerations are important when studying RBP7 as a prognostic biomarker in colon cancer?

RBP7 has emerged as an independent biomarker of poor cancer-specific survival in colon cancer. For robust biomarker studies:

• Regional sampling strategy:

  • Sample both tumor edge and center regions (≥100 μm from edge)

  • RBP7 expression is significantly higher at the tumor-stroma interface compared to tumor center

• Automated quantification:

  • Use QuPath or similar software for automated detection algorithms

  • Apply computational color deconvolution to separate stains

  • Calculate H-scores based on both staining intensity and frequency

• EMT pathway investigation:

  • Gene set enrichment analysis reveals strong association between RBP7, colon cancer invasion, and epithelial-mesenchymal transition (EMT)

  • Include EMT markers in parallel analyses

• Functional validation:

  • Complement expression studies with migration and invasion assays

  • Ectopic expression of RBP7 increases migration and invasion of colon cancer cells

• Long-term follow-up data:

  • Correlate RBP7 expression with both early and late-stage colon cancer outcomes

  • RBP7 is an independent biomarker of poor cancer-specific survival in both stages

How can I design experiments to investigate the relationship between RBP7 and PPARγ signaling in endothelial cells?

RBP7 functions as an endothelium-specific PPARγ cofactor. To study this relationship:

• PPARγ agonist/antagonist experiments:

  • Treat immortalized mouse lung endothelial cells (MLECs) with:

    • PPARγ agonist (rosiglitazone)

    • PPARγ antagonist (GW9662)

    • Combination treatment (pretreatment with antagonist followed by agonist)

  • Measure RBP7 mRNA and protein expression by qPCR and Western blot

• PPARγ overexpression:

  • Transfect cells with PPARγ expression vectors

  • Assess RBP7 induction with and without rosiglitazone stimulation

  • RBP7 protein is induced by either rosiglitazone or PPARγ overexpression, with enhanced expression when combined

• Target gene analysis:

  • Perform RNA sequencing to identify PPARγ target genes requiring RBP7

  • Focus on genes like adiponectin, which requires RBP7 for induction by rosiglitazone in endothelial cells

• Functional rescue experiments:

  • Test if adiponectin treatment rescues endothelial dysfunction in RBP7-deficient vessels

  • Measure endothelium-dependent relaxation and oxidative stress markers

For these experiments, include appropriate controls:

• Vehicle controls for drug treatments

• Empty vector controls for overexpression studies

• RBP7-deficient vs. control tissues/cells for comparative analyses

What are the critical considerations for Western blot protocols when detecting RBP7 protein?

Detecting RBP7 protein by Western blot requires specific technical considerations:

• Sample preparation:

  • Load 30 μg protein extracts on 15% SDS-PAGE gels (higher percentage gels are better for resolving low molecular weight proteins like RBP7)

  • Transfer to PVDF membranes for optimal protein binding

• Blocking conditions:

  • Block with either 10% skim milk for 30 minutes or

  • Use 4% non-fat dry milk in 1x TBST for overnight antibody incubation

• Antibody incubation:

  • Primary antibody dilution: 1:500-1:2000 for most RBP7 antibodies

  • Some protocols specifically recommend 1:2000 dilution for murine RBP7 antibodies

  • Incubate overnight at 4°C for optimal binding

• Detection parameters:

  • Expected molecular weight: 15.5 kDa calculated, 15-18 kDa observed

  • Use appropriate secondary antibody (HRP-linked anti-rabbit IgG at 1:5000 dilution for rabbit primary antibodies)

  • Develop with ECL plus reagents for optimal sensitivity

• Controls:

  • Positive control: HEK-293 cells, mouse adipose tissue

  • Loading control: β-Actin (1:10,000 dilution)

  • Validation control: Include samples with RBP7 knockdown or overexpression

How can I troubleshoot inconsistent RBP7 antibody staining in immunohistochemistry applications?

When facing inconsistent staining patterns with RBP7 antibodies in IHC:

• Antigen retrieval optimization:

  • Primary recommendation: Use TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Compare both methods to determine optimal conditions for your specific tissue

• Antibody concentration titration:

  • Test a range of dilutions (1:200-1:800)

  • Prepare a dilution series and test on consecutive sections of the same tissue

• Tissue-specific considerations:

  • For human colon cancer and kidney tissue, which are known to express RBP7, optimize protocols specifically for these tissues

  • Consider using tissue microarrays to test multiple conditions simultaneously

• Blocking optimization:

  • Test different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking time if background staining is high

• Signal amplification systems:

  • For weak signals, consider using polymer-based detection systems

  • Biotin-based systems may be appropriate for some applications, but can increase background

• Technical validation:

  • Include positive controls (tissue with confirmed RBP7 expression)

  • Run parallel staining with different RBP7 antibody clones or lots

  • Confirm specificity with a corresponding Western blot

What are the key considerations when using RBP7 antibodies for studying its role in retinoid metabolism?

When investigating RBP7's function in retinoid metabolism:

• Experimental model selection:

  • 3T3-L1 adipocytes are well-established models for studying RBP7's role in retinoid metabolism

  • Consider the differential expression of RBP7 during adipocyte differentiation (low at day 0, increasing by day 8)

• Complementary methodologies:

  • Combine protein expression analysis (Western blot) with functional assays (RARE-luciferase activity)

  • Assess both RBP7 and its downstream targets (LRAT, RALDH1, CYP26A1)

• Retinoic acid supplementation:

  • Include experiments with and without all-trans retinoic acid supplementation

  • Document time and dose-dependent effects

• Transcriptional activity measurement:

  • Use RARE-luciferase reporter assays to measure the transcriptional activity of retinoic acid receptors

  • RBP7 overexpression activates RARE, while knockdown tends to suppress it

• Feedback mechanisms:

  • Monitor expression of genes involved in:

    • Retinol storage (Lrat)

    • RA synthesis (Raldh1)

    • RA degradation (Cyp26a1)

  • These genes show distinct expression patterns in response to RBP7 modulation

• Antibody selection:

  • Choose antibodies against both RBP7 and retinoid metabolism proteins

  • For LRAT detection: Anti-LRAT (#PA5-38556, 1:1,000)

  • For ALDH1A1 detection: Anti-ALDH1A1 (#NB-100-787, 1:500)

  • For CYP26A1 detection: Anti-CYP26A1 (#PA5-24602, 1:1,000)