LSM5 Antibody

What is LSM5 and what is its function in cellular processes?

LSM5 (LSM5 Homolog, U6 Small Nuclear RNA Associated) is a protein involved in RNA processing pathways. It forms part of the LSM family that plays crucial roles in RNA metabolism, particularly in pre-mRNA splicing and mRNA degradation pathways. The protein has a molecular weight of approximately 12 kDa and functions within both nuclear and cytoplasmic LSM complexes. In the nucleus, LSM5 participates in the U6 snRNP complex for pre-mRNA splicing, while in the cytoplasm, it contributes to mRNA decay processes .

What types of LSM5 antibodies are available for research applications?

Researchers can access several types of LSM5 antibodies, including:

• Polyclonal antibodies: Generated in rabbit, goat, and other species, providing broad epitope recognition

• Monoclonal antibodies: Offering high specificity for particular epitopes

• Region-specific antibodies: Targeting different domains (C-terminal, N-terminal, middle region)

• Tagged/conjugated antibodies: Some preparations include fluorescent or enzymatic conjugates

Both polyclonal (e.g., rabbit anti-LSM5) and monoclonal (e.g., mouse anti-LSM5) antibodies are commercially available, with varying specificities for different regions of the LSM5 protein .

Which species reactivity is commonly available in LSM5 antibodies?

LSM5 antibodies demonstrate cross-reactivity across multiple species due to the highly conserved nature of this protein. Available antibodies typically react with:

• Human LSM5

• Mouse LSM5

• Rat LSM5

• Additional reactivity often includes: cow, guinea pig, horse, rabbit, dog, zebrafish, and in some cases, yeast (Saccharomyces cerevisiae)

This broad cross-reactivity (often 93-100% sequence homology) makes these antibodies versatile tools for comparative studies across different model organisms .

What are the validated applications for LSM5 antibodies in research?

LSM5 antibodies have been validated for multiple experimental applications:

• Western Blotting (WB): Detecting the ~12 kDa LSM5 protein in cell and tissue lysates

• Immunohistochemistry (IHC): Visualizing LSM5 in paraffin-embedded tissues

• Immunocytochemistry (ICC): Examining subcellular localization

• Immunofluorescence (IF): Determining cellular distribution patterns

• Flow Cytometry: Quantifying LSM5 expression in cell populations

• ELISA: Quantitative measurement of LSM5 levels

Researchers should note that specific antibody clones may be optimized for particular applications, so selecting the appropriate antibody for your experimental design is critical .

What are the optimal conditions for Western blot detection of LSM5?

For optimal Western blot detection of LSM5:

• Sample preparation: Use 50 μg of protein lysate under reducing conditions

• Gel selection: 5-20% SDS-PAGE is recommended for optimal separation

• Electrophoresis parameters: Run at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours

• Transfer conditions: Transfer to nitrocellulose membrane at 150mA for 50-90 minutes

• Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: 0.5-2 μg/ml of anti-LSM5 antibody, incubated overnight at 4°C

• Washing: TBS with 0.1% Tween, three times for 5 minutes each

• Secondary antibody: Anti-rabbit IgG-HRP at 1:5000 dilution for 1.5 hours at room temperature

• Detection: Enhanced chemiluminescence (ECL)

The expected band size for LSM5 is approximately 12 kDa. Specific cell lines showing reliable LSM5 detection include HL-60, K562, and MCF-7 .

How should LSM5 antibodies be used for immunohistochemistry and immunofluorescence?

For successful IHC and IF applications with LSM5 antibodies:

Immunohistochemistry protocol:

• Tissue preparation: Use paraffin-embedded sections

• Antigen retrieval: Heat-mediated retrieval in EDTA buffer (pH 8.0)

• Blocking: 10% goat serum

• Primary antibody: 2 μg/ml anti-LSM5 antibody, incubated overnight at 4°C

• Secondary antibody: Biotinylated anti-rabbit IgG, incubated for 30 minutes at 37°C

• Detection: Streptavidin-biotin complex with DAB as chromogen

Immunofluorescence protocol:

• Cell fixation: 4% paraformaldehyde

• Permeabilization: Use appropriate permeabilization buffer

• Blocking: 10% goat serum

• Primary antibody: 5 μg/ml anti-LSM5 antibody, incubated overnight at 4°C

• Secondary antibody: Fluorophore-conjugated anti-rabbit IgG (e.g., DyLight®488)

• Counterstain: DAPI for nuclear visualization

• Visualization: Fluorescence microscopy with appropriate filter sets

Both protocols have been validated in various tissues including cancer tissues (rectal, bladder, lung, ovary, renal, melanoma) and normal tissues like liver .

How can researchers minimize background and non-specific binding when using LSM5 antibodies?

To reduce background and enhance specificity:

• Optimization of antibody concentration: Titrate primary antibody (0.5-5 μg/ml range) to determine optimal signal-to-noise ratio

• Enhanced blocking: Extend blocking time to 2 hours or use alternative blocking agents (BSA, casein)

• Washing optimization: Increase wash duration or number of washing steps

• Antibody diluent modification: Add 0.1-0.2% Tween-20 to antibody diluent

• Secondary antibody selection: Use secondary antibodies pre-adsorbed against potential cross-reactive species

• Sample preparation: Ensure complete cell lysis and protein denaturation for Western blotting

• Negative controls: Include samples without primary antibody to evaluate secondary antibody specificity

For fluorescence applications, autofluorescence can be reduced using specific quenching agents or selecting fluorophores with excitation/emission profiles distinct from cellular autofluorescence .

What storage and handling precautions should be taken with LSM5 antibodies?

For optimal antibody performance and longevity:

• Long-term storage: Maintain at -20°C in small aliquots to prevent freeze-thaw cycles

• Short-term storage: For immediate use, store at 2-8°C for up to one week

• Reconstitution: For lyophilized antibodies, reconstitute according to manufacturer instructions

• Post-reconstitution stability: Store at 4°C for one month or re-aliquot and freeze at -20°C for up to six months

• Avoid repeated freeze-thaw cycles: These significantly reduce antibody activity

• Buffer considerations: Most antibodies are supplied in PBS with sodium azide (0.09%) and sucrose (2%)

• Safety precautions: Handle sodium azide-containing solutions with care as it is hazardous

Proper storage and handling ensure consistent antibody performance across experiments .

How can LSM5 antibodies be used to investigate RNA processing mechanisms?

For studying LSM5's role in RNA metabolism:

• Co-immunoprecipitation (Co-IP): Use LSM5 antibodies to pull down LSM5 protein complexes, followed by mass spectrometry or Western blotting to identify interaction partners

• RNA immunoprecipitation (RIP): Apply LSM5 antibodies to isolate LSM5-RNA complexes, coupled with sequencing to identify RNA targets

• Chromatin immunoprecipitation (ChIP): Investigate potential interactions between LSM5 and chromatin

• Proximity ligation assay (PLA): Visualize interactions between LSM5 and other proteins in situ

• FRET analysis: When using fluorescently-labeled antibodies, can detect protein-protein interactions

• Immunofluorescence co-localization: Determine subcellular co-localization with spliceosome components

These approaches help elucidate the functional role of LSM5 in splicing complexes and mRNA degradation pathways .

What considerations should be made when using LSM5 antibodies for flow cytometry?

For successful flow cytometry applications:

• Cell preparation: Fix cells with 4% paraformaldehyde and permeabilize using an appropriate buffer

• Blocking: Use 10% normal serum from the same species as the secondary antibody

• Antibody concentration: Typically 1 μg per 1×10^6 cells

• Controls: Include isotype controls and unstained samples

• Compensation: When using multiple fluorophores, perform proper compensation

• Gating strategy: Establish appropriate gates based on controls

• Data analysis: Analyze shifts in mean fluorescence intensity rather than just positive/negative gating

Flow cytometry has been validated with cell lines such as A431, showing successful detection of intracellular LSM5 .

How is LSM5 being investigated in cancer research?

LSM5 has emerging significance in cancer research:

• Expression analysis: LSM5 shows altered expression in multiple cancer types

• Immunohistochemical profiling: LSM5 antibodies have been used to examine LSM5 expression in:

  • Rectal cancer

  • Bladder cancer

  • Lung cancer

  • Ovarian cancer

  • Renal carcinoma

  • Melanoma

• Functional studies: Recent research has identified LSM5 as a potential biomarker for chemotherapy resistance, particularly in gastric cancer

• Immune infiltration correlation: Studies have found associations between LSM5 expression and immune cell infiltration, suggesting potential implications for immunotherapy responses

• Prognostic value: Research is exploring LSM5 as a prognostic indicator in various cancers

These findings position LSM5 as a protein of interest in cancer biology and potential therapeutic targeting .

What methodological approaches can be used to investigate LSM5's role in chemotherapy resistance?

To study LSM5 in chemotherapy resistance contexts:

• Expression correlation: Analyze LSM5 expression levels in relation to drug response using qPCR, Western blot, and IHC

• Gene silencing experiments: Use siRNA or CRISPR-Cas9 to knockdown LSM5 and assess changes in drug sensitivity

• Overexpression studies: Express LSM5 in sensitive cell lines to determine if resistance is conferred

• Patient-derived xenografts: Evaluate LSM5 expression in PDX models with varying drug responses

• Cell viability assays: Compare drug sensitivity between cells with different LSM5 expression levels

• RNA processing analysis: Investigate if LSM5-mediated RNA processing affects expression of drug resistance genes

• Immune correlation studies: Assess the relationship between LSM5 expression, immune cell infiltration, and therapy response

Research has specifically identified LSM5 as potentially related to 5-FU chemotherapy resistance in gastric cancer, suggesting it may serve as a biomarker for treatment response prediction .

How might novel LSM5 antibody developments enhance RNA biology research?

Emerging antibody technologies could advance LSM5 research:

• Single-domain antibodies: Nanobodies against LSM5 could improve imaging resolution and access to epitopes in complex structures

• Bifunctional antibodies: Dual-targeting antibodies could simultaneously detect LSM5 and interaction partners

• Intrabodies: Cell-penetrating antibodies could track LSM5 in living cells

• Degradation-targeting antibodies: PROTACs or similar technologies linked to LSM5 antibodies could enable precise protein degradation

• Epitope-specific antibodies: Development of antibodies recognizing specific LSM5 post-translational modifications

• Spatially-resolved antibody applications: Integration with spatial transcriptomics to correlate LSM5 localization with RNA processing events

These approaches could significantly enhance our understanding of LSM5's dynamic role in RNA metabolism under various cellular conditions .

What experimental approaches could best address the relationship between LSM5 and immune cell function in the tumor microenvironment?

To investigate LSM5's role in immune contexts:

• Multiplex immunohistochemistry: Simultaneously detect LSM5 and immune cell markers to analyze spatial relationships

• Single-cell RNA sequencing: Correlate LSM5 expression with immune cell subtypes and states

• Immune cell co-culture experiments: Study how modulating LSM5 in cancer cells affects immune cell behavior

• Cytokine profiling: Measure how LSM5 expression levels correlate with cytokine production

• Immune checkpoint correlation: Analyze associations between LSM5 and immune checkpoint molecules

• In vivo immune competent models: Study how LSM5 manipulation affects tumor-immune interactions in immunocompetent animals

• Patient sample analysis: Correlate LSM5 expression with immune infiltration and immunotherapy response

Recent research has begun exploring connections between LSM5 expression and immune cell infiltration in gastric cancer, suggesting potential implications for immunotherapy response prediction .