FABP4 Antibody, HRP conjugated

What is the optimal dilution range for FABP4 Antibody, HRP conjugated in Western blotting?

The optimal dilution range for HRP-conjugated FABP4 antibodies in Western blotting typically falls between 1:500 to 1:5000, depending on the specific antibody and manufacturer. For example, Abcam's anti-FABP4 HRP antibody [EPR3579] (ab216528) has been effectively used at 1:50 dilution for IHC-P applications . When determining the optimal dilution:

• Start with the manufacturer's recommended range

• Perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000)

• Evaluate signal-to-noise ratio at each dilution

• Select the dilution that provides clear detection of the ~15 kDa FABP4 band with minimal background

For Western blot applications, FABP4 antibodies detect a specific band at approximately 14-15 kDa under reducing conditions, as demonstrated in multiple validation studies .

What sample preparation techniques ensure optimal FABP4 detection?

Proper sample preparation is crucial for reliable FABP4 detection:

For tissue samples:

• Flash-freeze tissues immediately after collection

• Homogenize in RIPA buffer supplemented with protease inhibitors

• For adipose tissue, a modified extraction buffer containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) improves protein solubilization

For cell culture:

• For differentiated adipocytes, a specialized differentiation protocol is important as seen in 3T3-L1 studies

• Use differentiation stimulated medium composed of basal medium, 10% FBS, 50 μg/ml gentamicin, 1 nM L-glutamine, 500 μM IBMX, 1 μM dexamethasone, 2 μM rosiglitazone, and 1 μg/ml insulin

• Lyse cells directly in the plate with ice-cold lysis buffer

For immunohistochemistry:

• Use 10% neutral buffered formalin fixation for 24-48 hours

• For paraffin sections, heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0) for 20 minutes is recommended

• For frozen sections, acetone fixation for 10 minutes at -20°C preserves epitope recognition

How can background signal be reduced when using FABP4 Antibody, HRP conjugated in IHC?

High background is a common challenge with HRP-conjugated antibodies. The following optimization strategies can help:

Blocking optimization:

• Extend blocking time to 1-2 hours using 5% BSA or 5-10% normal serum

• Add 0.1-0.3% Triton X-100 to blocking buffer for better penetration

• Include 0.1% Tween-20 in wash buffers

Antibody incubation:

• Dilute antibody in blocking buffer containing 1-2% BSA

• Incubate at 4°C overnight rather than at room temperature

• Include additional wash steps (5-6 washes of 5 minutes each)

Endogenous peroxidase quenching:

• Treat sections with 0.3-3% hydrogen peroxide in methanol for 10-30 minutes

• For tissues with high endogenous peroxidase activity (e.g., adipose tissue), use commercial peroxidase blocking reagents

Documented example from literature:
When using ab216528 for IHC in human breast tissue, researchers achieved optimal results by pre-treating the section using heat-mediated antigen retrieval with sodium citrate buffer (pH6) for 20 minutes, followed by incubation with the antibody at 1:50 dilution for 15 minutes at room temperature .

What are the best practices for validating FABP4 antibody specificity?

Validating antibody specificity is crucial for reliable results:

Recommended validation approaches:

Practical example:
In validation studies, HRP-conjugated FABP4 antibodies showed strong staining in the cytoplasm of human bladder cancer tissue and adipose tissue, while showing minimal background in negative control sections without primary antibody incubation .

How can FABP4 Antibody, HRP conjugated be used to study the relationship between adipocyte dysfunction and metabolic disease?

FABP4 antibodies serve as valuable tools for investigating adipocyte function in metabolic disorders:

Experimental approach:

• Use HRP-conjugated FABP4 antibody to quantify expression changes in:

  • Diet-induced obesity models

  • Insulin resistance progression

  • Inflammatory states in adipose tissue

• Dual staining protocols:

  • Co-stain for FABP4 and inflammatory markers (TNF-α, IL-6)

  • Assess FABP4 co-localization with insulin signaling components

  • Evaluate FABP4 expression in crown-like structures in obese adipose tissue

Research findings:
Studies have identified that FABP4 plays a crucial role in the development of insulin resistance and atherosclerosis in relation to metaflammation. FABP4 has been directly associated with cardiac alterations such as left ventricular hypertrophy (LVH) and both systolic and diastolic cardiac dysfunction .

Supporting this, knockout studies in mice have shown that FABP4-deficient mice fed a high-fat and high-calorie diet become obese but develop neither insulin resistance nor diabetes, suggesting this protein might be a link between obesity and insulin resistance .

What considerations are important when using FABP4 antibodies in multiplex immunofluorescence studies?

When designing multiplex studies with FABP4 antibodies:

Antibody panel design:

• Select antibodies raised in different host species to avoid cross-reactivity

• If using multiple rabbit antibodies, consider sequential staining with tyramide signal amplification

• Ensure spectral separation between fluorophores

Documented multiplex application:
Immunofluorescence analysis of co-cultures of HDLECs and C2C12 cells successfully labeled FABP4 and CD31 using a rabbit anti-FABP4 antibody (green) at 10 μg/ml and a mouse anti-CD31 monoclonal antibody (red), detected with appropriate fluorophore-conjugated secondary antibodies .

Optimization considerations:

• Titrate each antibody individually before combining

• Test for and eliminate cross-reactivity between antibodies

• Include appropriate single-stain controls for spectral unmixing

• Consider the sequence of antibody application (lower abundance targets first)

How does HRP-conjugated FABP4 antibody compare to unconjugated antibodies for various applications?

The choice between HRP-conjugated and unconjugated FABP4 antibodies depends on the specific application requirements:

Research findings:
For Western blot applications, paired antibody testing has demonstrated that unconjugated anti-FABP4 antibodies can detect FABP4 protein in both human and mouse samples at dilutions of 1:500, while HRP-conjugated versions may require different optimization parameters .

What methodological differences should be considered when using recombinant versus conventional FABP4 antibodies?

The production method significantly impacts antibody performance:

Recombinant antibodies (e.g., Abcam's EPR3579 clone):

• Offer superior batch-to-batch consistency

• Eliminate need for same-lot requests

• Provide highly reproducible results across experiments

• Typically have higher specificity

Conventional polyclonal antibodies:

• May recognize multiple epitopes on FABP4

• Can show batch variability

• Sometimes offer better sensitivity for certain applications

• May be advantageous when protein conformation is altered

The recombinant monoclonal antibody clone EPR3579 has been documented as the most widely used clone for FABP4 on the market, with specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation .

How should Western blot quantification of FABP4 be standardized for comparative studies?

Proper quantification of FABP4 in Western blot requires standardization:

Recommended quantification protocol:

• Use 15% SDS-PAGE to efficiently resolve the 14-15 kDa FABP4 protein

• Include recombinant FABP4 standards (ranging from 0.1-100 ng) for calibration curve

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

• Use appropriate housekeeping proteins for normalization:

  • β-actin for general normalization

  • Adipocyte-specific markers for adipose tissue samples

• Quantify band intensities using digital image analysis software

• Express results as:

  • Ratio of FABP4 to housekeeping protein

  • Absolute quantification using the standard curve

Demonstrated example:
Studies using 3T3-L1 differentiation models have successfully quantified FABP4 expression changes by separating 20 μg of whole cell extracts by 15% SDS-PAGE, blotting with FABP4 antibody diluted at 1:500, and detecting with HRP-conjugated anti-rabbit IgG antibody .

How can contradictory FABP4 expression data between different detection methods be reconciled?

Researchers occasionally encounter discrepancies in FABP4 expression between methods:

Reconciliation strategies:

• Epitope accessibility differences:

  • Western blot detects denatured proteins

  • IHC/ICC detects proteins in native conformation

  • Solution: Use multiple antibodies recognizing different epitopes

• Sensitivity thresholds:

  • ELISA typically offers higher sensitivity than Western blot

  • Solution: Use appropriate dilution series for each method

• Sample preparation variations:

  • Fixation can mask epitopes in IHC

  • Solution: Compare different fixation and antigen retrieval methods

• Quantification approaches:

  • Western blot provides semi-quantitative data

  • ELISA provides absolute quantification

  • Solution: Generate standard curves for each method

Case study:
Validation studies have demonstrated that the same anti-FABP4 antibody can detect FABP4 in multiple applications with different optimal conditions: for Western blot analysis, lysates of human and mouse heart tissue at 0.2 mg/mL showed specific bands at approximately 19 kDa, while in IHC, the same antibody at 3 μg/mL successfully detected FABP4 in human bladder cancer tissue sections .

What considerations are important when designing experiments to study FABP4's role in disease mechanisms?

When investigating FABP4's role in disease pathology:

Key experimental design elements:

• Model selection:

  • Cell models: 3T3-L1 adipocytes, primary adipocytes, macrophage cell lines

  • Animal models: Diet-induced obesity, genetic models (db/db, ob/ob), FABP4 knockout models

  • Human samples: Adipose tissue biopsies, serum samples for circulating FABP4

• Intervention approaches:

  • Pharmaceutical inhibition of FABP4

  • siRNA/shRNA knockdown

  • CRISPR/Cas9 gene editing

  • Recombinant FABP4 treatment

• Endpoint measurements:

  • FABP4 expression (protein and mRNA)

  • Metabolic parameters (glucose tolerance, insulin sensitivity)

  • Inflammation markers

  • Lipid composition analysis

Research context:
Studies have identified FABP4 as potentially therapeutic target for treating metabolic disorders. Mice carrying a FABP4 genetic variant exhibit both reduced FABP4 expression and reduced potential for developing type 2 diabetes and coronary heart disease. Similar patterns have been observed in human studies, suggesting FABP4 may be a potential therapeutic target in the treatment of these disorders .

How should time course experiments be designed to study FABP4 regulation during adipogenesis?

Time course experiments require careful planning:

Recommended experimental design:

• Cell model preparation:

  • Use preadipocyte cell lines (3T3-L1, 3T3-F442A) or primary stromal vascular cells

  • Ensure cells reach appropriate confluence (70-80%)

• Differentiation protocol:

  • Induce differentiation with standard cocktail (IBMX, dexamethasone, insulin, rosiglitazone)

  • Sample collection at key timepoints:

    • Day 0 (pre-induction)

    • Day 2 (early commitment)

    • Day 4 (intermediate differentiation)

    • Day 7 (advanced differentiation)

    • Day 10 (mature adipocytes)

• Analysis methods:

  • Western blot with HRP-conjugated FABP4 antibody

  • qRT-PCR for FABP4 mRNA

  • Immunofluorescence for cellular localization changes

  • Oil Red O staining to correlate with lipid accumulation

Validated approach:
In 3T3-L1 differentiation studies, researchers successfully used FABP4 antibodies to detect expression changes during adipogenesis. The differentiation stimulated medium composed of basal medium, 10% FBS, 50 μg/ml gentamicin, 1 nM L-glutamine, 500 μM IBMX, 1 μM dexamethasone, 2 μM rosiglitazone, and 1 μg/ml insulin effectively induced FABP4 expression that could be monitored using appropriate antibodies .