TSKU Antibody, FITC conjugated

What is TSKU Antibody, FITC conjugated, and what are its primary research applications?

TSKU antibody, FITC conjugated, is a polyclonal antibody that targets specific amino acid sequences (commonly AA 231-300) of human Tsukushin (TSKU/LRRC54) protein. The antibody is derived from rabbit hosts and is directly conjugated with fluorescein isothiocyanate (FITC), which emits green fluorescence when excited at 499nm.

Primary applications include:

• Immunofluorescence on cultured cells (IF-cc)

• Immunofluorescence on paraffin-embedded sections (IF-p)

• Immunofluorescence on frozen tissue sections (IF-f)

• Flow cytometry for cell surface or intracellular staining

For optimal results, recommended dilutions typically range from 1:50-1:200 depending on the specific application, with the following guidelines :

What are the optimal storage conditions for maintaining TSKU antibody, FITC conjugated activity?

To maintain optimal activity of FITC-conjugated TSKU antibodies, follow these evidence-based storage guidelines:

• Store at -20°C in small aliquots to avoid repeated freeze-thaw cycles

• Protect from light (FITC is photosensitive)

• Store in buffer containing stabilizers (typically PBS pH 7.4 with 50% glycerol)

• Include protein stabilizers such as BSA (1-5 mg/mL) to prevent non-specific binding

• Some preparations include 0.03% Proclin-300 as a preservative instead of sodium azide

How can I optimize TSKU antibody, FITC conjugated staining protocols for different cell types?

Optimization of FITC-conjugated TSKU antibody staining requires adjustments based on cell type, fixation method, and target localization:

For adherent cells (immunocytochemistry):

• Culture cells on coverslips and fix with 4% paraformaldehyde (15 min, RT)

• Permeabilize with 0.1% Triton X-100 if detecting intracellular epitopes

• Block with 5% normal serum from the same species as secondary antibody

• Incubate with TSKU antibody-FITC at 1:50-1:100 dilution (overnight, 4°C)

• Wash extensively with PBS (3×10 min)

• Counterstain nuclei with DAPI if desired

• Mount with anti-fade mounting medium

For tissue sections:

• For paraffin sections: Include antigen retrieval steps (typically citrate buffer pH 6.0 or EDTA buffer pH 8.0)

• For frozen sections: Fix briefly (5 min) to preserve morphology while maintaining epitope accessibility

Critical optimization parameters:

• Antibody concentration (titrate between 1:25-1:200)

• Incubation time and temperature

• Blocking reagents (use species-matched normal serum)

• Washing stringency

What are common issues with FITC-conjugated antibodies and how can they be resolved?

Issue 1: High background fluorescence

• Cause: Non-specific binding or autofluorescence

• Solution: Increase blocking time/concentration (5-10% serum), include 0.1-0.3% Triton X-100 in blocking buffer

• Advanced approach: Include unconjugated, isotype-matched control antibodies in blocking solution

Issue 2: Weak or fading signal

• Cause: Photobleaching of FITC, suboptimal F/P ratio, or improper storage

• Solution: Minimize exposure to light, use anti-fade mounting media containing DABCO or n-propyl gallate

• Data: Studies show that anti-fade reagents can extend FITC fluorescence lifetime by 5-10× under continuous illumination

Issue 3: Signal heterogeneity across samples

• Cause: Inconsistent conjugation or variable epitope accessibility

• Solution: Use site-specific conjugation methods like transglutaminase-mediated labeling to ensure consistent F/P ratios

• Technical approach: Consider deglycosylation using PNGase F followed by site-specific conjugation as described by Chung et al.

How can I verify the specificity of TSKU antibody, FITC conjugated in my experimental system?

Comprehensive validation of TSKU antibody specificity requires multiple complementary approaches:

• Positive and negative control tissues/cells:

  • Positive: Tissues known to express TSKU (based on RNA-seq databases)

  • Negative: TSKU-knockout cells or tissues with confirmed absence of expression

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide (10-100× molar excess)

  • Specific binding should be blocked by peptide competition

• Orthogonal validation:

  • Confirm TSKU expression using alternative methods (qPCR, Western blot)

  • Use multiple antibodies targeting different TSKU epitopes (e.g., AA 77-353 vs. AA 231-300)

• siRNA knockdown:

  • Transfect cells with TSKU-specific siRNA

  • Specific staining should be diminished proportionally to knockdown efficiency

How can site-specific conjugation improve TSKU antibody-FITC performance compared to traditional conjugation methods?

Traditional FITC conjugation uses amine-reactive chemistry, resulting in heterogeneous labeling of random lysine residues. Site-specific conjugation offers several scientifically proven advantages:

Comparison of conjugation methods:

Site-specific conjugation approaches for FITC labeling:

• Transglutaminase method:

  • Deglycosylate antibody using PNGase F to expose Gln295

  • Use microbial transglutaminase (MTGase) to attach FITC-cadaverine

  • Results in consistent 1:1 labeling ratio with preserved antibody function

• Click chemistry approach:

  • Introduce azide-handles to antibody using enzymatic reactions

  • React with DBCO-modified FITC using strain-promoted azide-alkyne cycloaddition

  • Allows precise control of conjugation site and stoichiometry

Experimental data shows site-specific conjugates exhibit 50-100% higher binding activity retention compared to randomly labeled antibodies, with significant improvements in signal-to-noise ratios .

What advanced imaging techniques are most compatible with TSKU antibody, FITC conjugated?

FITC-conjugated TSKU antibodies can be leveraged in multiple advanced imaging platforms:

Super-resolution microscopy:

• Structured Illumination Microscopy (SIM): FITC is well-suited with ~100nm resolution

• Stimulated Emission Depletion (STED): Requires careful optimization of imaging parameters

• Photoactivated Localization Microscopy (PALM): Not ideal as FITC lacks the required photoswitching properties

Intravital microscopy:

• Two-photon microscopy: FITC can be excited at ~800nm for deeper tissue penetration

• Limitations: FITC photobleaches more rapidly than newer fluorophores in vivo

Correlative Light and Electron Microscopy (CLEM):

• FITC signals can be preserved through specialized fixation protocols

• DAB photooxidation can convert FITC fluorescence to electron-dense precipitates

Multiplexed imaging considerations:

• FITC (Ex/Em: 499/515nm) pairs well with red fluorophores (e.g., Cy3, Alexa594)

• Minimal spectral overlap with far-red dyes (Cy5, Alexa647)

• Consider spectrally distinct nuclear counterstains (e.g., DAPI or propidium iodide)

How can I conduct co-localization studies using TSKU antibody, FITC conjugated with other cellular markers?

Rigorous co-localization studies require careful experimental design and quantitative analysis:

Experimental protocol:

• Select complementary markers with spectrally distinct fluorophores

  • Membrane markers: Anti-CD44-Cy3 for cell surface co-localization

  • Vesicular markers: Anti-LAMP1-Alexa647 for lysosomal trafficking

• Perform sequential or simultaneous immunostaining based on host species compatibility

• Include single-stained controls for spectral unmixing and bleed-through correction

• Acquire images with identical settings across all samples

Quantitative co-localization analysis:

• Pearson's correlation coefficient (PCC): Values from -1 (negative correlation) to +1 (positive correlation)

• Manders' overlap coefficient: Proportion of TSKU signal overlapping with second marker

• Object-based methods: Count percentage of TSKU+ structures also positive for second marker

Advanced approaches:

• Live cell imaging with FITC-conjugated TSKU antibody fragments to track trafficking

• Fluorescence resonance energy transfer (FRET) to detect molecular proximity (<10nm)

• Light sheet microscopy for rapid 3D co-localization in thick samples

What is known about TSKU protein function and how can FITC-conjugated antibodies contribute to its investigation?

TSKU (Tsukushin) is a secreted protein belonging to the small leucine-rich proteoglycan (SLRP) family. Key functions include:

• Modulation of signaling pathways (BMP, Wnt, Notch)

• Extracellular matrix organization

• Potential roles in development and disease processes

FITC-conjugated TSKU antibodies enable several functional investigations:

• Internalization and trafficking studies:

  • Pulse-chase experiments to track TSKU endocytosis

  • Co-localization with markers of endocytic pathways

  • Similar to experimental designs used for HER2 antibody internalization studies

• Binding competition assays:

  • Determine whether TSKU antibodies compete with natural ligands

  • Identify functional binding domains through epitope-specific antibodies

• Live cell dynamics:

  • Using humanized or directly-conjugated Fab fragments

  • Time-lapse studies of TSKU membrane distribution and clustering

• Proximity-based interaction studies:

  • Combined with proximity ligation assays (PLA) to detect TSKU-interaction partners

  • FRET-based approaches when paired with suitable acceptor fluorophores

How does FITC compare to other fluorophores for TSKU antibody conjugation in various research applications?

Different fluorophores offer distinct advantages depending on the research application:

FITC limitations include:

• Higher photobleaching rate compared to Alexa dyes

• pH sensitivity (fluorescence decreases below pH 7)

• Smaller Stokes shift (increasing risk of autofluorescence interference)

For advanced applications requiring maximum sensitivity or photostability, consider using site-specific conjugation with Alexa488 instead of FITC, which can provide 2-3× higher signal-to-noise ratios in challenging samples.

How do different site-specific conjugation methods compare for creating FITC-labeled TSKU antibodies?

Various site-specific conjugation technologies offer different advantages for creating homogeneous FITC-labeled antibodies:

Research by Chung et al. demonstrates that click chemistry approaches result in functional antibody conjugates with no significant aggregation compared to traditional methods . The CovIsolink platform utilizing Q-tag technology showed consistent drug-antibody ratios of 1.7 for full IgGs, with preserved binding affinity to target receptors .

What are emerging applications for TSKU antibody, FITC conjugated in biomedical research?

Several cutting-edge applications are being developed:

• Single-cell analysis platforms:

  • Mass cytometry (CyTOF) using metal-conjugated TSKU antibodies

  • Spatial transcriptomics combined with FITC-immunofluorescence

• Microfluidic systems:

  • Antibody-functionalized microchannels for rare cell capture

  • Droplet-based single-cell analysis with FITC immunostaining

• Biosensor development:

  • FRET-based biosensors using FITC-TSKU antibodies paired with acceptor fluorophores

  • Label-free detection systems using TSKU antibody fragments

• Therapeutic applications:

  • TSKU-targeted antibody-drug conjugates (ADCs)

  • CAR-T cell development using TSKU-targeting domains

• 3D model systems:

  • Organoid imaging to track TSKU expression during development

  • Tissue clearing techniques compatible with FITC-conjugated antibodies

How might advances in site-specific conjugation further improve TSKU antibody applications?

Future advancements in site-specific conjugation technologies are likely to benefit TSKU antibody applications in several ways:

• Enhanced control over orientation:

  • Directing FITC away from antigen-binding regions

  • Optimizing accessibility of binding domains

• Dual-labeled antibodies:

  • Precisely positioned FRET pairs for conformational studies

  • Antibodies with both detection and functional moieties

• Homogeneous antibody fragments:

  • Site-specifically labeled Fab, scFv, and VHH constructs

  • Data indicates DAR values of 0.8 can be consistently achieved for antibody fragments using Q-tag technology

• Format chain exchange technology (FORCE):

  • Generation of binder-format-payload matrices for optimized conjugates

  • Enables rapid screening of different antibody formats with consistent conjugation

• Clinical translation:

  • More homogeneous conjugates with improved pharmacokinetics

  • Reduced batch-to-batch variability for diagnostic applications