RASAL2 Antibody, FITC conjugated

What is RASAL2 and what is its biological significance?

RASAL2 (RAS protein activator like 2), also known as nGAP, is a Ras GTPase-activating protein containing a GRD domain and a coiled-coil structure at the C terminus . It functions as a regulator of the Ras signaling pathway, influencing processes including tumorigenesis, epithelial-mesenchymal transition, and tumor metastasis . Recent research has identified RASAL2 as a metabolic regulator involved in energy homeostasis and adipogenesis . The protein is encoded by the RASAL2 gene (Gene ID: 9462) and has a calculated molecular weight of 142 kDa with 1262 amino acids .

RASAL2 expression is significantly increased in the livers of high-fat diet (HFD)-fed mice and in free fatty acid (FFA)-treated hepatocytes, suggesting its involvement in nonalcoholic fatty liver disease (NAFLD) . Microarray analysis from the GSE48452 dataset confirmed elevated RASAL2 expression in NAFLD patients compared to nonsteatotic controls .

What applications are RASAL2 antibodies suitable for?

RASAL2 antibodies have been validated for multiple research applications:

For FITC-conjugated RASAL2 antibodies specifically, optimal applications include flow cytometry, immunofluorescence microscopy, and live cell imaging. When designing experiments with FITC-conjugated antibodies, consider that FITC has excitation/emission peaks at approximately 495/519 nm, placing it in the green fluorescence spectrum.

How should RASAL2 antibodies be validated for experimental use?

Proper validation involves multiple complementary approaches:

• Genetic validation: Use RASAL2-deficient models as negative controls. Both RASAL2 knockout mice and RASAL2 knockdown cell lines have been successfully employed to confirm antibody specificity .

• Molecular weight verification: Confirm detection of bands at the expected molecular weight (140-150 kDa for the Proteintech antibody or 90 kDa for the Abbexa antibody ).

• Positive controls: HeLa cells have been validated for RASAL2 expression and serve as effective positive controls . Human liver tissues from NAFLD patients show elevated RASAL2 expression compared to nonsteatotic controls .

• Signal titration: Test multiple antibody dilutions to determine optimal signal-to-noise ratio for your specific application and sample type.

• Cross-reactivity assessment: Evaluate potential cross-reactivity with related proteins, particularly other Ras GTPase-activating proteins.

What storage and handling protocols maximize RASAL2 antibody performance?

For optimal performance of RASAL2 antibodies, including FITC-conjugated versions:

• Store at -20°C in manufacturer-recommended buffer conditions

• Some formulations are stable for one year after shipment when stored properly

• Avoid repeated freeze/thaw cycles to prevent degradation

• For FITC-conjugated antibodies, protect from light exposure to prevent photobleaching

• The Proteintech antibody is supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• The Abbexa antibody requires reconstitution in 100 μl of sterile distilled H₂O with 50% glycerol

How does FITC conjugation impact RASAL2 antibody performance in different applications?

FITC conjugation can affect antibody performance in several key ways:

What methodological approaches optimize RASAL2 detection in hepatic steatosis models?

Based on recent research on RASAL2's role in hepatic steatosis , several methodological considerations are important:

• Model selection:

  • In vivo: RASAL2-deficient mice (Rasal2PB/PB) versus wild-type littermates on high-fat diet provide an effective model system .

  • In vitro: L02 and HepG2 cells treated with 1 mM free fatty acid mixture (oleic acid:palmitic acid=2:1) serve as cellular models .

• Essential readouts:

  • Lipid accumulation assessment via Oil Red O staining, BODIPY493/503 staining, and triglyceride quantification

  • VLDL secretion rate determination via tyloxapol injection

  • Protein expression analysis of RASAL2, phospho-AKT, TET1, and MTTP via western blotting

• Pathway analysis protocols:

  • Investigate the AKT/TET1/MTTP axis through selective inhibitor treatments (e.g., triciribine for AKT inhibition)

  • Perform 5-hmC immunoprecipitation to assess DNA hydroxymethylation of the MTTP gene promoter

  • Use chromatin immunoprecipitation to confirm TET1 binding to the MTTP promoter region

• Antibody application strategies:

  • For immunohistochemistry of liver sections, use dilutions of 1:100-1:200

  • For western blot detection of RASAL2 upregulation in steatotic livers, use dilutions of 1:500-1:3000

  • When using FITC-conjugated antibodies for immunofluorescence, increase antibody concentration to compensate for potential signal reduction

What controls are essential when using FITC-conjugated RASAL2 antibodies in immunofluorescence studies?

Rigorous control strategies for immunofluorescence studies with FITC-conjugated RASAL2 antibodies include:

• Biological controls:

  • Positive tissue controls: HeLa cells have been validated for RASAL2 expression

  • Negative tissue controls: RASAL2-deficient models show minimal to no specific staining

  • Expression gradient samples: Compare normal liver tissue versus steatotic liver, which shows upregulated RASAL2 expression

• Technical controls:

  • Autofluorescence control: Unstained sample to establish baseline tissue autofluorescence in the FITC channel

  • Isotype control: FITC-conjugated non-specific IgG from same host species and at the same concentration

  • Secondary antibody control: For two-step detection methods

  • Blocking validation: Pre-absorption with immunizing peptide if available

• Imaging controls:

  • Exposure standardization: Maintain identical exposure settings across all comparable samples

  • Spectral unmixing: When multiplexing with other fluorophores

  • Photobleaching assessment: Repeated imaging of the same field to establish FITC signal stability

How can RASAL2 antibodies be used to investigate the AKT/TET1/MTTP pathway in metabolic research?

The AKT/TET1/MTTP pathway represents a novel mechanism by which RASAL2 regulates hepatic lipid metabolism . Methodological approaches for investigating this pathway include:

• Pathway characterization:

  • Use western blotting with RASAL2 antibodies to establish baseline expression in different metabolic states

  • Monitor phosphorylated AKT levels as RASAL2 deficiency increases AKT phosphorylation

  • Track changes in TET1 expression and MTTP upregulation following RASAL2 manipulation

• Mechanism dissection:

  • Employ selective inhibitors (e.g., triciribine for AKT inhibition) to identify critical pathway components

  • Perform TET1 knockdown experiments to validate its role in mediating RASAL2 effects

  • Measure global 5-mC and 5-hmC levels to assess epigenetic changes

• Functional assessment protocols:

  • Measure VLDL secretion rates to evaluate the functional outcome of pathway activation

  • Quantify triglyceride accumulation to assess the metabolic impact of pathway modulation

  • Analyze insulin sensitivity parameters (FBG, FBI, HOMA-IR) to evaluate broader metabolic effects

Research has shown that RASAL2 deficiency activates the AKT pathway, leading to increased TET1 expression, which promotes MTTP expression through DNA hydroxymethylation, ultimately increasing VLDL production and reducing hepatic lipid accumulation .

What troubleshooting approaches address common challenges with FITC-conjugated RASAL2 antibodies?

When encountering difficulties with FITC-conjugated RASAL2 antibodies, consider these methodological solutions:

• Weak signal issues:

  • Increase antibody concentration (starting with 2-3 fold higher than unconjugated versions)

  • Extend incubation time (e.g., overnight at 4°C)

  • Use signal amplification systems compatible with FITC (e.g., anti-FITC antibodies)

  • Optimize fixation protocols, as overfixation can mask epitopes

  • Implement antigen retrieval methods for tissue sections

• High background challenges:

  • Increase blocking stringency (5-10% serum from species unrelated to antibody host)

  • Add 0.1-0.3% Triton X-100 to antibody diluent to reduce non-specific binding

  • Perform longer washing steps (5-6 washes of 5 minutes each)

  • Include 0.05% Tween-20 in wash buffers

  • Use Sudan Black B (0.1%) treatment to reduce tissue autofluorescence

• Photobleaching management:

  • Minimize exposure during imaging

  • Use anti-fade mounting media containing DABCO or similar compounds

  • Consider sequential imaging strategies for multicolor applications

  • Adjust imaging settings to balance signal capture with photobleaching

• Specificity verification:

  • Compare results with unconjugated RASAL2 antibodies

  • Validate using RASAL2-deficient models as negative controls

  • Confirm correct subcellular localization patterns

How do experimental conditions affect RASAL2 expression and detection?

RASAL2 expression and detection are significantly influenced by experimental conditions:

• Diet-induced metabolic changes:

  • High-fat diet feeding significantly increases hepatic RASAL2 protein levels in mice

  • This elevation occurs progressively over 12 weeks of HFD feeding

• Lipid loading effects:

  • Treatment of hepatocytes with 1 mM free fatty acid mixture (oleic acid:palmitic acid=2:1) increases RASAL2 expression in vitro

  • This cellular model replicates the in vivo findings from high-fat diet experiments

• Tissue-specific expression patterns:

  • Immunohistochemistry analysis shows significant elevation of RASAL2 in steatotic human livers compared to nonsteatotic controls

  • These findings are consistent with microarray data from the GSE48452 dataset

• Antibody detection considerations:

  • The observed molecular weight can vary between antibodies (140-150 kDa for Proteintech vs. 90 kDa for Abbexa )

  • Sample preparation methods can affect epitope accessibility

  • Positive controls such as HeLa cells should be included to validate detection methods

These findings highlight the importance of carefully controlled experimental conditions when studying RASAL2 in metabolic research and suggest that RASAL2 antibodies can serve as valuable tools for monitoring metabolic stress responses.