IFITM2 Antibody

What are the optimal applications for IFITM2 antibodies in viral research?

IFITM2 antibodies are valuable tools for investigating viral entry mechanisms and antiviral responses. They are particularly useful for:

• Western blot analysis to detect IFITM2 protein expression (15-17 kDa observed molecular weight) in various cell lines including HeLa, MCF7, HepG2, and A549

• Immunohistochemistry of paraffin-embedded tissues, particularly effective in human lung cancer tissue

• Immunofluorescence microscopy to examine subcellular localization of IFITM2, which shows distribution in both endolysosomal compartments and the plasma membrane

• Immunoprecipitation studies to investigate protein interactions and post-translational modifications

Recommended dilutions vary by application:

Note: Always optimize antibody concentrations for specific experimental conditions and sample types .

How can I differentiate between IFITM2 and other IFITM family members when using antibodies?

Distinguishing between IFITM family members presents a significant challenge due to high sequence homology. Key approaches include:

• Use isoform-specific antibodies: Select antibodies raised against unique regions of IFITM2, particularly those targeting the N-terminal domain where sequence divergence is greatest

• Employ knockout/knockdown controls: Include IFITM2-specific knockdown samples to validate antibody specificity

• Cross-reactivity assessment: Most commercial antibodies for IFITM2 have cross-reactivity with IFITM3 due to their highly conserved protein sequences (approximately 91% sequence identity)

• Molecular weight discrimination: IFITM2 has an observed molecular weight of 15-17 kDa, which can sometimes be distinguished from other family members by careful Western blot analysis

For unambiguous identification, combine antibody detection with molecular techniques such as RT-PCR using isoform-specific primers targeting unique regions of the IFITM2 transcript .

What are the critical considerations for immunofluorescence detection of IFITM2 subcellular localization?

For accurate subcellular localization studies of IFITM2:

• Fixation method selection: For standard fluorescence microscopy, acetone/methanol (1:1) fixation for 5 minutes on ice is recommended; for confocal microscopy, use 4% paraformaldehyde for 10 minutes followed by 0.1% Triton X-100 permeabilization for 10 minutes

• Blocking conditions: Use 10% normal goat serum for 30 minutes at room temperature to reduce nonspecific binding

• Primary antibody incubation: Optimal dilutions range from 1:50-1:500 in 1% BSA, incubated for 1 hour at room temperature or overnight at 4°C

• Co-staining markers: Include markers for specific compartments to accurately determine localization:

  • Rab5a for early endosomes

  • Rab7 for late endosomes

  • Lamp1 for lysosomes

Research has shown that Δ20 IFITM2 (lacking 20 amino acids at the N-terminus) displays a distinct localization pattern compared to full-length IFITM2, with ring distribution at the plasma membrane in addition to endolysosomal compartments .

How should antigen retrieval be optimized for IFITM2 immunohistochemistry in different tissue types?

Optimal antigen retrieval for IFITM2 immunohistochemistry varies by tissue type:

• Epithelial tissues (lung, breast): Heat-induced epitope retrieval using citrate buffer (pH 6.0) under high pressure is recommended. For lung cancer tissue, this approach with a 1:700 dilution of IFITM2 antibody showed excellent results

• Alternative method for difficult tissues: TE buffer (pH 9.0) can improve antigen retrieval in tissues with high protein crosslinking

• Detection system selection: An HRP-conjugated SP system provides optimal visualization after incubation with a biotinylated secondary antibody

For paraffin-embedded tissue sections:

• Dewax and hydrate sections

• Perform antigen retrieval with appropriate buffer based on tissue type

• Block with 10% normal goat serum for 30 minutes at room temperature

• Incubate with primary antibody (1% BSA) overnight at 4°C

• Apply biotinylated secondary antibody and visualize using an HRP detection system

How can IFITM2 antibodies be utilized to investigate differential restriction of X4 versus R5 HIV-1 by Δ20 IFITM2?

Investigating the selective restriction of X4 (CXCR4-tropic) but not R5 (CCR5-tropic) HIV-1 by Δ20 IFITM2 requires specialized experimental approaches:

• Expression analysis: Use antibodies recognizing both isoforms to determine relative expression of full-length IFITM2 versus Δ20 IFITM2 in CD4+ T cells, monocytes, and dendritic cells. Research shows Δ20 IFITM2 is highly expressed in these immune cells and is the predominant isoform compared to full-length IFITM2

• Subcellular distribution assessment: Perform immunofluorescence to determine Δ20 IFITM2 localization at both plasma membrane and endolysosomal compartments, which is critical for understanding its selective restriction mechanisms

• Viral entry assays: Use pseudotyped X4 and R5 HIV-1 in conjunction with IFITM2 immunoblotting to correlate expression levels with restriction activity:

• Receptor interaction studies: Combine immunoprecipitation with receptor expression analysis to determine whether Δ20 IFITM2 affects CD4, CXCR4, or CCR5 surface expression or distribution

For validation, knockdown experiments in primary cells have shown that depletion of IFITM2 enhances infection of X4 but not R5 HIV-1 in monocyte-derived dendritic cells and macrophages .

What methodological approaches are needed to evaluate the role of IFITM2 in SARS-CoV-2 entry using specific antibodies?

Recent research has revealed that SARS-CoV-2 uniquely hijacks IFITM2 for efficient infection, contrary to the typical antiviral role of IFITM proteins. Key methodological approaches include:

• Specific antibody selection: Utilize antibodies targeting the N-terminal extracellular domain of IFITM2, such as the 5D11B9 monoclonal antibody that specifically blocks SARS-CoV-2 entry

• Spike protein internalization assays: Monitor the effect of anti-IFITM2 antibodies on Spike-mediated endocytosis using fluorescently labeled Spike protein

• Cell fusion assays: Quantify syncytia formation in the presence/absence of anti-IFITM2 antibodies to assess membrane fusion events

• Cytopathic effect measurements: Compare viral cytopathology with and without IFITM2 antibody treatment

Multiple viral systems can be evaluated using this approach:

These findings suggest IFITM2 may represent a common pathway exploited by multiple viruses, offering potential broad-spectrum antiviral strategies targeting this host factor .

How can cross-reactivity issues between IFITM2 and IFITM3 antibodies be addressed in experimental settings?

Cross-reactivity between IFITM2 and IFITM3 antibodies presents a significant challenge due to their high sequence homology. Researchers can implement several strategies to address this issue:

• Transcript-specific analysis: Use RT-PCR with primers targeting unique regions (particularly the transcripts ENST00000399817 for full-length IFITM2 and ENST00000602569 for Δ20 IFITM2) to complement protein detection

• Epitope mapping: Select antibodies raised against regions with the greatest sequence divergence between IFITM2 and IFITM3

• Recombinant protein controls: Include purified recombinant IFITM2 and IFITM3 proteins as positive controls to establish specific banding patterns

• Knockout validation: Use CRISPR/Cas9-generated IFITM2 or IFITM3 knockout cells to confirm antibody specificity

• Differential expression systems: Utilize cell systems with differential expression of IFITM family members to characterize antibody specificity profiles

Western blot analysis typically shows:

• IFITM2: 15-17 kDa band

• IFITM3: Slightly higher molecular weight band that may overlap with IFITM2

For immunofluorescence applications, subcellular distribution patterns may help distinguish IFITM2 (both endolysosomal and plasma membrane) from IFITM3 (predominantly endolysosomal) .

What are the optimal conditions for detecting IFITM2 expression changes following interferon stimulation?

Detection of interferon-induced changes in IFITM2 expression requires careful experimental design:

• Time course considerations: IFITM2 expression peaks approximately 16-24 hours post-interferon stimulation

• Interferon type selection: Type I interferons (particularly IFN-α at 1000 units/ml) effectively induce IFITM2 expression

• Cell type selection: Expression patterns vary across cell types; CD4+ T cells, monocyte-derived dendritic cells, and macrophages show robust IFITM2 induction following IFN treatment

• Isoform-specific detection: Monitor both full-length and Δ20 IFITM2 expression, as the Δ20 isoform is the predominant form in immune cells

• Control treatments: Include phytohemagglutinin (PHA) controls, as PHA treatment has been shown to deplete IFITM2 expression, contrary to IFN effects

For optimal Western blot detection:

• Stimulate cells with IFN-α (1000 units/ml) for 16-24 hours

• Harvest cells in buffer containing phosphatase inhibitors to preserve post-translational modifications

• Resolve proteins on 12-15% gels to adequately separate the 15-17 kDa IFITM2 protein

• Transfer to PVDF membrane and probe with IFITM2-specific antibody at optimized dilution (1:2000-1:10000)

Research demonstrates that both full-length and Δ20 IFITM2 are upregulated by interferon stimulation, but their relative expression levels and subcellular distribution may differ depending on cell type and activation status .

How should researchers interpret differing results between IFITM2 antibodies in viral restriction assays?

When encountering discrepancies in results using different IFITM2 antibodies in viral restriction studies, consider these analytical approaches:

• Epitope targeting analysis: Determine if antibodies recognize different epitopes that might be differentially accessible in various experimental conditions or affected by protein conformation

• Isoform specificity: Evaluate whether antibodies differentially recognize full-length IFITM2 versus the Δ20 IFITM2 isoform, which have distinct antiviral activities

• Cell type considerations: Results may vary based on cell type due to differences in IFITM2 expression, post-translational modifications, or interacting partners. For example, U87 cells show different IFITM2-mediated restriction patterns compared to T cells

• Viral entry pathway influence: The impact of IFITM2 varies depending on viral entry mechanisms:

Research has revealed that contradictory results in HIV-1 restriction studies stemmed from differential activity of IFITM2 isoforms against X4 versus R5 viruses and varying expression patterns of these isoforms across cell types .

What experimental approach should be used to study the differential effects of IFITM2 on various viral families?

To comprehensively investigate IFITM2's impact across viral families, implement a multi-faceted experimental design:

• Viral panel selection: Include representatives from diverse viral families with different entry mechanisms:

  • Enveloped viruses: Influenza, SARS-CoV-2, HIV, HSV

  • Non-enveloped viruses: As negative controls

  • Viruses with different entry pathways: Direct fusion vs. endocytosis

• Combinatorial assays:

  • Entry assays using pseudotyped viruses expressing various viral glycoproteins

  • Replication assays measuring viral load in IFITM2 wildtype vs. knockout/knockdown cells

  • Membrane fusion assays to assess specific steps in viral entry

• IFITM2 variant analysis: Compare full-length IFITM2 and Δ20 IFITM2, as they show differential restriction patterns

Recent findings demonstrate varied IFITM2 effects across viruses:

The combination of targeted antibodies against specific IFITM2 domains with viral restriction assays has revealed that IFITM2 can either restrict or promote viral infection depending on the virus type and specific isoform involved .