FIS1 Antibody

What is FIS1 and what cellular processes does it participate in?

FIS1 (Fission 1) is an integral mitochondrial outer membrane protein that participates in mitochondrial fission by interacting with dynamin-related protein 1 (Drp1). It plays a critical role in regulating mitochondrial morphology. Excessive mitochondrial fission has been associated with several neurodegenerative and neurodevelopmental disorders, including Huntington's disease, Alzheimer's disease, and autism spectrum disorder . FIS1 functions as a recruitment factor for proteins like TBC1D15, which are involved in Drp1-independent regulation of mitochondrial morphology .

What are the validated applications for FIS1 antibody in research?

FIS1 antibody has been validated for multiple research applications including:

The antibody has been successfully employed in knockout/knockdown validation studies across 11 publications, confirming its specificity .

What are the recommended dilutions for different experimental applications?

The following dilutions are recommended for optimal results:

It is strongly recommended that researchers titrate the antibody in each specific testing system to achieve optimal signal-to-noise ratio .

How should I design co-immunoprecipitation experiments to study FIS1 protein interactions?

For effective co-immunoprecipitation of FIS1 and its binding partners:

• Solubilization conditions: Use digitonin (1%) rather than stronger detergents to preserve protein-protein interactions. The lysis buffer should contain 50 mM Tris-HCl pH 7.5, 150 mM NaCl, and protease inhibitor cocktails .

• Recommended procedure:

  • Clear lysates by centrifugation before immunoprecipitation

  • For tagged proteins, use monoclonal FLAG or polyclonal HA antibodies with protein-G–Sepharose or protein-A–Sepharose, respectively

  • For endogenous FIS1, use 0.5-4.0 μg of FIS1 antibody per 1.0-3.0 mg of total protein lysate

  • Analyze precipitants by SDS-PAGE followed by immunoblotting

• Control experiments: Always include appropriate negative controls such as IgG controls or cells not expressing the tagged proteins to confirm specificity of interactions .

What techniques are effective for studying FIS1's interaction with TBC1D15?

Based on published studies, the following approaches have proven effective:

• Co-immunoprecipitation: Express HA-tagged TBC1D15 with FLAG-tagged FIS1 in cells, then immunoprecipitate using either anti-FLAG or anti-HA antibodies after digitonin solubilization. This approach has confirmed direct interaction between these proteins .

• Pull-down assays: Use recombinant proteins like GST-tagged TBC1D15 fragments (particularly residues 200-300) and His-tagged FIS1. After bacterial co-expression, purify using glutathione-Sepharose beads in Tris-HCl buffer containing 150 mM NaCl, 5% glycerol, 1% Triton X-100, and 1 mM DTT. This method has demonstrated direct physical interaction between the proteins .

• Deletion analysis: To map interaction domains, create deletion mutants of TBC1D15 and co-express with FIS1. The region between residues 220-250 of TBC1D15 contains essential information for mitochondrial localization, while the minimal region required for FIS1-dependent recruitment is residues 240-300 .

How can I optimize immunofluorescence protocols for detecting endogenous FIS1?

For optimal detection of endogenous FIS1 by immunofluorescence:

• Fixation: Standard paraformaldehyde (4%) fixation works well for FIS1 detection

• Dilution optimization: Start with 1:200-1:800 dilution range and titrate for your specific cell type

• Mitochondrial co-localization: Use mitochondrial markers like MitoTracker or co-staining with other mitochondrial proteins to confirm localization

• Knockdown controls: Include FIS1-knockdown cells as negative controls to verify specificity, as FIS1-RNAi cells show clear release of TBC1D15 to the cytoplasm from mitochondria

• Cell types: The antibody has been validated in multiple cell lines including HeLa, Hepa1-6, and HepG2 cells

How can I use structural analysis to understand FIS1 protein dynamics?

For researchers investigating FIS1 structural dynamics:

• Molecular dynamics (MD) simulations:

  • Use 1000-ns simulations to evaluate possible conformations of the FIS1 arm

  • Multiple starting structures should be employed to avoid bias (e.g., PDB ID: 1PC2 and homology models)

  • Monitor Cα RMSD values, which typically stabilize after ~200 ns

  • MD simulations can reveal that the FIS1 arm may adopt an intramolecular conformation regardless of the starting structure

• NMR chemical shift analysis:

  • Compare experimental NMR chemical shifts to identify conformational differences

  • Use kernel density plot analyses to distinguish structural differences from those caused by buffer conditions

  • Pay special attention to the FIS1 arm region, which shows significant conformational dynamics

• Structural comparison methods:

  • Compare human FIS1 with mouse FIS1 structures to identify conserved features

  • Calculate all atom-atom distances to quantify conformational states (IN vs. OUT conformations)

What approaches can be used to study FIS1's role in disease models?

FIS1 has been implicated in several diseases, particularly neurodegenerative conditions:

• Disease model systems:

  • Huntington's disease (HD)-affected brain tissue

  • Alzheimer's disease (AD) patient samples

  • Autism spectrum disorder models

• Expression analysis techniques:

  • Western blot analysis of patient vs. control tissue (1:2000-1:14000 dilution)

  • Immunohistochemistry of brain tissue sections (1:50-1:500 dilution with TE buffer pH 9.0 for antigen retrieval)

  • Comparative expression across different brain regions and disease stages

• Mitochondrial morphology assessment:

  • Use immunofluorescence to correlate FIS1 expression with mitochondrial fragmentation

  • Combine with Drp1 staining to evaluate recruitment to mitochondria

  • Knockdown studies to determine the effect of FIS1 reduction on disease phenotypes

How should I interpret differences in FIS1 localization between knockdown conditions?

When analyzing FIS1 and its binding partner localization:

• FIS1 knockdown effects:

  • In control cells, TBC1D15 partially colocalizes with mitochondrial markers

  • In FIS1-RNAi cells, mitochondrial TBC1D15 is released to the cytoplasm

  • This confirms FIS1's role as a TBC1D15 receptor on mitochondria

• Comparison with other knockdowns:

  • Drp1 knockdown does not affect TBC1D15 localization

  • Mff knockdown does not affect TBC1D15 localization

  • FIS1 knockdown does not affect Drp1 foci on mitochondria

  • TBC1D15 knockdown does not affect Drp1 foci on mitochondria

• Interpretation: These results indicate that FIS1 functions specifically as a TBC1D15 receptor in the Drp1-independent regulation of mitochondrial morphology, rather than being involved in the mitochondrial recruitment of Drp1 .

What factors might affect the detection of the correct molecular weight for FIS1?

When analyzing FIS1 by Western blot:

• Expected molecular weight: FIS1 has a calculated molecular weight of 17 kDa, which is typically observed on Western blots .

• Sample preparation factors:

  • Incomplete denaturation can affect migration

  • Post-translational modifications might alter apparent molecular weight

  • Sample buffer composition (reducing vs. non-reducing) can affect results

• Positive control samples: The antibody has been validated in multiple sample types including:

  • HEK-293 cells, HeLa cells, Jurkat cells, HepG2 cells, PC-12 cells

  • Mouse/rat brain tissue, heart tissue, and spleen tissue

  • Using these validated samples as positive controls can help confirm proper detection

How can I verify the specificity of FIS1 antibody in my experimental system?

To ensure antibody specificity:

• Knockout/knockdown validation:

  • Include FIS1 knockout or knockdown controls

  • The antibody has been validated in 11 knockout/knockdown studies

• Multiple detection methods:

  • Compare results across different applications (WB, IF, IHC)

  • Consistent results across methods support antibody specificity

• Protein-protein interaction verification:

  • Confirm known interactions, such as with TBC1D15

  • Use co-immunoprecipitation followed by Western blot to verify

  • Pull-down assays with recombinant proteins can provide additional validation

How can FIS1 antibody be used to study mitochondrial fission mechanisms?

For mitochondrial dynamics research:

• Visualizing mitochondrial networks:

  • Use immunofluorescence (1:200-1:800 dilution) to detect FIS1 localization

  • Co-stain with other fission/fusion proteins (Drp1, Mff) to analyze recruitment patterns

  • Analyze changes in FIS1 distribution under different cellular stresses

• Interaction studies:

  • Use co-immunoprecipitation to identify novel FIS1 binding partners

  • Employ digitonin (1%) for membrane protein solubilization

  • Study how these interactions change under different physiological conditions

• Functional studies:

  • Combine with mitochondrial morphology analysis

  • Correlate FIS1 expression levels with fission events

  • Use in knockdown/knockout experiments to determine the essential nature of FIS1 in different fission pathways

The wealth of published studies using FIS1 antibody (380+ publications across various applications) provides a strong foundation for researchers to build upon when designing new experiments to investigate mitochondrial dynamics and related disease mechanisms .