GDF3 Antibody, HRP conjugated

What are the validated research applications for GDF3 antibodies?

GDF3 antibodies have been validated for multiple research applications including Western blotting (WB), immunohistochemistry (IHC), and immunocytochemistry/immunofluorescence (ICC-IF). According to validation data, rabbit polyclonal antibodies against GDF3 have demonstrated specific detection in all three applications . Recombinant monoclonal antibodies like EPR4828 show particular specificity in Western blotting applications with human samples . When designing experiments, researchers should select antibodies with validation data specifically matching their intended application and target species.

What tissues and cell types express significant levels of GDF3 for use as positive controls?

GDF3 expression has been confirmed in several specific tissues and cell types that serve as excellent positive controls:

• Mouse thymus (validated by immunohistochemistry)

• Human embryonic stem cells (BG01V line)

• Human neuroblastoma cells (SH-SY5Y)

• Human embryonic kidney cells (293T)

• Human fetal brain tissue

• Human hepatocellular carcinoma cells (HepG2)

• Human seminoma tissue

When establishing new GDF3 detection protocols, including at least one of these tissues or cell types as a positive control is highly recommended to validate antibody functionality.

What are the optimal dilutions and conditions for detecting GDF3 using antibodies in Western blotting?

For Western blot applications:

• Primary antibody: Recombinant monoclonal antibodies (e.g., EPR4828) show optimal results at 1/1000 dilution

• Sample loading: 10 μg of total protein lysate per lane is typically sufficient

• Secondary antibody: HRP-conjugated anti-rabbit IgG can be used at 1/2000-1/8000 dilution depending on the specific antibody sensitivity and signal strength required

• Expected band size: The mature GDF3 protein should be detected at approximately 41-42 kDa

• Reducing conditions: Standard reducing conditions with Immunoblot Buffer Group 1 have been validated for GDF3 detection

Include both positive control lysates (e.g., SH-SY5Y or 293T) and negative controls to ensure specificity of detection.

What is the recommended protocol for GDF3 immunohistochemical detection?

For immunohistochemical detection of GDF3:

• Tissue preparation: Both perfusion-fixed frozen sections and paraffin-embedded tissues have been successfully used

• Primary antibody concentration: 5-15 μg/mL for frozen sections (optimal incubation overnight at 4°C)

• For paraffin-embedded tissues: 1/25 dilution has been validated with human thyroid cancer tissue

• Detection system: Anti-Goat HRP-DAB Cell & Tissue Staining Kit works effectively with goat polyclonal GDF3 antibodies

• Counterstaining: Hematoxylin provides good nuclear contrast without obscuring cytoplasmic GDF3 staining

Always include a negative control by omitting the primary antibody to confirm specificity of the signal.

How can researchers study GDF3's role in sepsis and inflammation experimentally?

GDF3 has emerged as a protective factor in sepsis with therapeutic potential. To study this function:

• In vivo sepsis model:

  • Cecal ligation and puncture (CLP) model has been validated for studying GDF3 in sepsis

  • Recombinant GDF3 administration: 20 μg/kg body weight (preventive study, 3h before CLP) or 100 μg/kg body weight (therapeutic study, 1h after CLP) via tail vein injection

  • Parameters to measure:

    • Survival rate (monitor every 6h for 7 days)

    • Bacterial burden in blood and peritoneal lavage fluid (20-24h post-CLP)

    • Cytokine levels and markers of organ injury

• In vitro macrophage studies:

  • Bone marrow-derived macrophages (BMDMs) treated with recombinant GDF3 (20-50 ng/mL)

  • RAW264.7 macrophage cell line can also be used as an alternative model

  • Incubation of cells with rGDF3 for 18h prior to LPS stimulation (10 ng/mL)

  • For mechanism studies, include inhibitors like SB431542 (10 μM) to block ALK4/5/7 signaling upstream of Smad2/3

What molecular mechanisms mediate GDF3's anti-inflammatory effects, and how can they be investigated?

GDF3 exerts anti-inflammatory effects through specific signaling pathways that can be experimentally studied:

• Smad2/3 pathway analysis:

  • Measure Smad2/3 phosphorylation by Western blot following GDF3 treatment

  • Utilize SB431542 (ALK4/5/7 inhibitor) to confirm pathway specificity

  • Analyze NLRP3 expression, which is inhibited by GDF3 through Smad signaling

• Macrophage polarization assessment:

  • Flow cytometry to quantify M1 (pro-inflammatory) vs. M2 (anti-inflammatory) macrophage markers following GDF3 treatment

  • qRT-PCR for characteristic M1/M2 marker genes

  • RNA sequencing to identify broader transcriptional changes (CD5L and Bcl2a1b are GDF3-regulated genes of interest)

• LXRα-CD5L signaling examination:

  • Immunofluorescence staining for LXRα nuclear translocation

  • Combine GDF3 treatment with LXR agonists (GW3965, 1 μmol/L) or antagonists (GSK2033, 2 μmol/L)

  • qRT-PCR to measure CD5L and Bcl2a1b expression changes

What are the advantages and limitations of using HRP-conjugated secondary antibodies for GDF3 detection?

Advantages:

• High sensitivity: HRP-conjugated detection systems provide excellent signal amplification for detecting proteins with low expression levels

• Versatility: Compatible with multiple substrates (ECL, DAB) for different visualization methods

• Stability: HRP-conjugated antibodies typically maintain activity during proper storage

• Cost-effectiveness: Secondary antibody approach allows one HRP-conjugated reagent to be used with multiple primary antibodies

Limitations:

• Potential high background: May require optimization of blocking and washing steps

• Two-step detection: Requires additional incubation time compared to direct detection methods

• Species cross-reactivity: Must select secondary antibodies with appropriate host species specificity

What protocols can optimize signal-to-noise ratio when using HRP-conjugated antibodies with anti-GDF3 primaries?

To optimize signal-to-noise ratio:

• Validated dilution ratios:

  • Primary anti-GDF3 antibody: 1/250-1/1000 dilution range works for most applications

  • HRP-conjugated secondary: 1/2000-1/8000 dilution range, with 1/8000 showing good results with properly optimized systems

• Exposure time optimization:

  • Start with short exposures (1 minute) and adjust as needed

  • Avoid prolonged exposure which can increase background

• Sample preparation considerations:

  • For Western blots: 40 μg of tissue lysate or 10 μg of cell line lysate provides sufficient protein for detection

  • 6% SDS-PAGE gels have been validated for GDF3 detection

How can researchers assess GDF3's impact on bacterial phagocytosis and killing by macrophages?

Multiple validated protocols exist for studying GDF3's effect on macrophage antimicrobial functions:

• Phagocytosis assay:

  • Treat peritoneal macrophages with BSA (control) or rGDF3 (20 ng/mL) for 18 hours

  • Challenge with pHrodo red E. coli BioParticles for 1.5 hours at 37°C

  • Analyze by flow cytometry after fixing cells with 2% paraformaldehyde

• Bactericidal activity assessment:

  • Pre-treat BMDMs or RAW264.7 cells with rGDF3 (20 ng/mL) for 18 hours

  • Infect with live E. coli at MOI of 20:1 (bacteria:cell) for 1 hour

  • Add gentamicin (100 μg/mL) for 30 minutes to kill extracellular bacteria

  • Lyse cells and plate for CFU counting at 1 hour (phagocytosis) and 6 hours (killing) timepoints

  • Calculate killing percentage: [(CFU at 1h) - (CFU at 6h)]/(CFU at 1h) × 100%

This methodological approach provides quantitative data on both phagocytic capacity and bacterial killing efficiency.

What high-throughput approaches can identify novel GDF3-regulated genes in macrophages?

RNA sequencing has successfully identified GDF3-regulated genes in macrophages:

• Experimental design:

  • Treat BMDMs with BSA (control) or rGDF3 (20 ng/mL)

  • Extract RNA and perform high-throughput RNA sequencing

  • Create heatmaps of differentially expressed genes

• Validation approach:

  • Confirm key differentially expressed genes (e.g., CD5L, Bcl2a1b, MRPS18C) by qRT-PCR

  • Compare gene regulation patterns with known agonists/antagonists of related pathways (e.g., LXR pathway modulators)

• Functional correlation:

  • Connect transcriptional changes to phenotypic effects (e.g., CD5L upregulation correlates with enhanced phagocytosis)

  • Use immunofluorescence to visualize transcription factor translocation (e.g., LXRα) that may drive these transcriptional changes

What are the most common sources of false results in GDF3 detection and how can they be addressed?

• Non-specific bands in Western blotting:

  • Use validated antibodies with demonstrated specificity (e.g., EPR4828)

  • Include positive control lysates with known GDF3 expression (SH-SY5Y, 293T, or fetal brain)

  • Verify band size matches the expected 41-42 kDa for GDF3

  • Optimize blocking conditions and antibody dilutions to reduce background

• Background staining in immunohistochemistry:

  • Always include a negative control without primary antibody

  • Use antigen affinity-purified antibodies to increase specificity

  • Optimize antibody concentration (5-15 μg/mL range for frozen sections)

  • Select tissues with confirmed GDF3 expression (e.g., thymus) as positive controls

• Inter-laboratory variability:

  • Standardize protocols using the validated conditions reported in literature

  • Document lot numbers of antibodies and reagents used

  • Consider recombinant monoclonal antibodies for higher batch-to-batch consistency