SWI5 Antibody, FITC conjugated

What is SWI5 protein and what cellular functions does it perform?

SWI5 (SWI5 Recombination Repair Homolog) is a protein that functions as a component of the SWI5-SFR1 complex, which is required for double-strand break repair via homologous recombination . This protein plays a critical role in maintaining genomic stability by facilitating DNA repair processes. The human SWI5 gene is also known as C9orf119 and is identified by UniProt ID Q1ZZU3 . Understanding SWI5's function is essential when designing experiments to investigate DNA repair mechanisms, particularly in cancer research and genomic stability studies.

What does FITC conjugation mean and how does it benefit antibody applications?

FITC (fluorescein isothiocyanate) conjugation refers to the chemical linking of the fluorescent dye FITC to an antibody molecule. FITC is not simply used as a fluorescent dye; like biotin and DIG, it is used for labeling proteins and nucleic acid probes . The conjugation process involves the isothiocyanate reactive group (-N=C=S) binding to primary amines on the antibody. This allows direct visualization of the antibody-antigen interaction through fluorescence microscopy without requiring secondary detection methods. FITC emits green fluorescence when excited with blue light, making it suitable for various fluorescence-based applications including flow cytometry, immunofluorescence microscopy, and ELISA .

What is the target specificity of commercially available SWI5-FITC antibodies?

The commercially available SWI5 antibodies with FITC conjugation typically target amino acids 54-112 of the human SWI5 protein . These antibodies are generally raised in rabbits and are polyclonal in nature, meaning they recognize multiple epitopes within this amino acid region . The specificity is confirmed through recombinant human DNA repair protein SWI5 homolog protein (54-112AA) as the immunogen . This specific binding region ensures reliable detection of the SWI5 protein in various experimental applications while minimizing cross-reactivity with other proteins.

How should optimal dilutions be determined for different experimental applications?

Determining optimal dilutions for SWI5-FITC antibodies requires systematic titration experiments for each application type. As noted in product documentation, "Optimal working dilution should be determined by the investigator" . A recommended approach is to perform a dilution series (typically starting from 1:100 to 1:5000) in the specific application of interest. For ELISA, prepare a standard curve with known concentrations of recombinant SWI5 protein and test multiple antibody dilutions to identify the concentration that provides the best signal-to-noise ratio. For immunofluorescence applications, test various dilutions on positive control samples (cells or tissues known to express SWI5) alongside negative controls. The optimal dilution should provide clear specific staining with minimal background fluorescence.

What buffer systems and storage conditions maximize SWI5-FITC antibody performance?

SWI5-FITC antibodies are typically supplied in a liquid form containing 0.01M PBS at pH 7.4 with 0.03% Proclin-300 and 50% glycerol as stabilizers . For long-term storage, aliquot the antibody and store at -20°C or -80°C to avoid repeated freeze-thaw cycles that can degrade antibody performance . It's critical to protect FITC-conjugated antibodies from light exposure as FITC is photosensitive and can photobleach, reducing fluorescence intensity over time . For working solutions, dilute the antibody in fresh buffer containing 1-5% BSA or suitable blocking protein. If stored at 2-8°C after reconstitution, use within one month; for longer storage (up to 6 months), keep at -20°C to -70°C under sterile conditions .

How can signal sensitivity be enhanced when using SWI5-FITC antibodies in low-expression samples?

When working with samples where SWI5 expression is low, several signal amplification strategies can be employed. One approach is to use an anti-FITC antibody system for signal enhancement. FITC-labeled proteins can be directly observed, but their detection sensitivity can be significantly increased by using an anti-FITC antibody followed by a labeled secondary antibody . This creates a layered detection system that amplifies the original signal. Alternative approaches include using tyramide signal amplification (TSA) systems compatible with FITC fluorescence or employing more sensitive detection instruments with higher-quality filters and detectors. Combining these approaches with optimized sample preparation methods, such as antigen retrieval for fixed tissues, can further enhance detection capabilities in challenging samples.

What controls should be included when designing experiments with SWI5-FITC antibodies?

Rigorous experimental design for SWI5-FITC antibody applications should include multiple control types:

These controls help distinguish true positive signals from artifacts, particularly important when investigating the SWI5-SFR1 complex in DNA repair pathways where specific localization patterns may be subtle or transient following DNA damage induction.

How can co-localization studies be designed to investigate SWI5 interactions with other DNA repair proteins?

Co-localization studies examining SWI5 interactions with other repair proteins require careful planning of multiplexed immunofluorescence experiments. Since the SWI5 antibody is already conjugated to FITC (green fluorescence), select complementary fluorophores for other target proteins that have minimal spectral overlap, such as rhodamine/Cy3 (red) or Cy5 (far-red). Design the staining protocol to include appropriate blocking steps between primary antibodies if using the same host species. For quantitative co-localization analysis, capture high-resolution z-stack images using confocal microscopy and analyze using specialized software that calculates Pearson's correlation coefficient or Mander's overlap coefficient. Consider including DNA damage induction (e.g., ionizing radiation or chemical agents) to observe dynamic recruitment of SWI5 and partner proteins to damage sites, which would provide functional insights into the temporal aspects of complex formation during the DNA repair process.

What are common causes of high background when using SWI5-FITC antibodies in immunofluorescence?

High background in immunofluorescence experiments with SWI5-FITC antibodies can stem from several sources:

• Insufficient blocking: Increase blocking time or concentration of blocking reagent (5% BSA or normal serum)

• Excessive antibody concentration: Perform additional titration experiments to find optimal dilution

• Autofluorescence from fixatives: Consider using different fixation methods; aldehyde-based fixatives can increase autofluorescence

• Photobleaching of FITC: Minimize exposure to light during all experimental steps

• Proclin-300 in antibody buffer: The preservative can sometimes contribute to background; consider buffer exchange if problematic

Implementing a step-wise troubleshooting approach by modifying one variable at a time will help identify and resolve the specific cause of background issues in your experimental system.

How should results be interpreted when studying SWI5 localization during different phases of DNA damage response?

Interpreting SWI5 localization patterns during DNA damage response requires understanding its normal distribution and damage-induced changes. Under basal conditions, SWI5 typically shows diffuse nuclear localization. Following DNA damage, expect redistribution to discrete nuclear foci, representing sites of double-strand breaks where homologous recombination repair occurs. The kinetics of this relocalization are important—early timepoints (minutes to 2 hours post-damage) might show initial recruitment, while later timepoints (4-24 hours) could show resolution as repair completes. Quantification should include both the number of SWI5-positive foci per nucleus and their colocalization with established DNA damage markers (γH2AX, RAD51). Cell cycle-specific analysis is crucial since homologous recombination is predominantly active in S and G2 phases. Incorporate cell cycle markers to distinguish cell cycle-dependent variations in SWI5 distribution from damage-specific responses.

What strategies can address cross-reactivity issues with SWI5-FITC antibodies?

To address potential cross-reactivity issues with SWI5-FITC antibodies, implement these methodological approaches:

• Validate with genetic controls: Use SWI5 knockout/knockdown cells alongside wild-type cells to confirm specificity

• Perform peptide competition assays: Pre-incubate the antibody with excess immunizing peptide (AA 54-112) to block specific binding

• Compare results with alternative antibody clones: Use antibodies targeting different SWI5 epitopes to corroborate findings

• Implement Western blot validation: Confirm a single band of appropriate molecular weight before proceeding with more complex applications

• Use orthogonal detection methods: Validate findings with non-antibody methods (e.g., fluorescent protein tagging of SWI5)

When interpreting results in complex samples, consider the similarity between SWI5 and other DNA repair proteins, particularly those containing similar structural domains, and include appropriate controls to distinguish specific from non-specific signals.

How can SWI5-FITC antibodies be used to study DNA repair dynamics in response to different genotoxic agents?

SWI5-FITC antibodies can be leveraged to investigate differential DNA repair responses to various genotoxic agents through time-course experiments. Design studies comparing ionizing radiation (generating mainly double-strand breaks) versus UV radiation or chemical agents like methyl methanesulfonate (producing other lesion types). For live-cell imaging applications, combine the antibody with cell-permeable DNA damage markers and perform microinjection of the SWI5-FITC antibody. Alternatively, for fixed-cell analysis, treat cells with different agents, fix at defined timepoints (15min, 30min, 1h, 4h, 24h), and perform immunostaining. Quantify the number, size, and intensity of SWI5-positive repair foci using high-content imaging systems. Advanced analysis should include colocalization with pathway-specific markers (RAD51 for homologous recombination, 53BP1 for non-homologous end joining) to determine how SWI5 recruitment differs between repair pathways activated by different damage types.

What methodological approaches can distinguish between SWI5 individual functions versus its role in the SWI5-SFR1 complex?

Distinguishing between individual SWI5 functions and its role in the SWI5-SFR1 complex requires sophisticated experimental designs:

• Proximity ligation assay (PLA): Use SWI5-FITC antibody with anti-SFR1 antibody to visualize only the complexed form of SWI5

• Co-immunoprecipitation: Perform IP with SWI5 antibody followed by Western blotting for SFR1 under different conditions

• FRET analysis: If using additional fluorophore-labeled antibodies against SFR1, measure FRET efficiency to assess protein-protein proximity

• Mutant complementation studies: Express SWI5 mutants defective in SFR1 binding but retaining other functions

• Chromatin fractionation: Compare the chromatin-bound versus soluble pools of SWI5 with and without DNA damage

These approaches will help determine whether observed phenotypes result from SWI5's role in the complex or from potential independent functions, particularly important when interpreting results from cancer cells where complex formation may be dysregulated.

How can quantitative image analysis be optimized for SWI5-FITC signal in complex tissue microenvironments?

Quantitative image analysis of SWI5-FITC signals in complex tissues requires addressing several technical challenges:

• Spectral unmixing: Implement computational algorithms to separate FITC signal from tissue autofluorescence, particularly pronounced in FFPE tissues

• Segmentation strategies: Use nuclear markers (DAPI) for primary segmentation, followed by subcellular compartmentalization

• Machine learning approaches: Train algorithms to recognize true SWI5 foci versus artifacts based on size, shape, and intensity parameters

• 3D analysis methods: For tissue sections, acquire z-stacks and perform 3D reconstruction to avoid false negatives from out-of-plane foci

• Internal normalization: Include reference cells or beads with known fluorescence intensity to standardize measurements across samples

These advanced imaging methodologies enable more accurate quantification of SWI5 expression and localization patterns in heterogeneous tissue samples, crucial for translational studies examining DNA repair capacity in tumor versus normal tissues.