RPS4Y1 Antibody, HRP conjugated

What is RPS4Y1 and why is it significant for gender-specific cellular research?

RPS4Y1 (40S ribosomal protein S4, Y isoform 1) is a protein encoded by the Y-linked gene RPS4Y1 located on Yp11.31. This protein is exclusively expressed in male cells and tissues, making it an ideal male-specific marker. The significance of RPS4Y1 lies in its early expression during embryonic development, immediately after embryonic genome activation at the E3 stage (8-cell stage), and its continued expression throughout development .

The protein shows high homology (approximately 93% sequence identity) with its X-chromosome counterpart, RPS4X, which is expressed in both males and females . Despite this homology, specific antibodies can distinguish between these two proteins, making RPS4Y1 antibodies valuable tools for gender determination at the cellular level.

In research settings, RPS4Y1 antibodies are particularly valuable for:

• Detecting male cells in mixed cell populations

• Isolating fetal cells from maternal blood for non-invasive prenatal diagnosis

• Studying X-linked inherited diseases such as hemophilia

How does RPS4Y1 antibody specificity overcome cross-reactivity with RPS4X?

The main challenge in developing specific RPS4Y1 antibodies stems from the high sequence homology between RPS4Y1 and RPS4X proteins (93% identity) . To achieve specificity, researchers have identified unique epitope regions within the RPS4Y1 sequence that differ from RPS4X.

A successful approach documented in research involves targeting three small regions (Y1, Y2, Y3) with the highest number of amino acid differences specific to the RPS4Y1 protein:

• Y1: 17 amino acids with 2 RPS4Y1-specific residues

• Y2: 16 amino acids with 3 RPS4Y1-specific residues

• Y3: 23 amino acids with 4 RPS4Y1-specific residues

Of these regions, the 155-177 amino acid region of RPS4Y1 has been identified as particularly immunogenic and suitable for antibody production . Antibodies targeting this region have demonstrated high specificity for RPS4Y1 without cross-reactivity with RPS4X, as confirmed by Western blotting analyses using male (HepG2) and female (HEK293) cell lysates .

What are the standard experimental applications for RPS4Y1 antibody, HRP conjugated?

RPS4Y1 antibody, HRP conjugated, can be utilized in multiple experimental techniques:

The HRP conjugation provides direct enzymatic detection capabilities, eliminating the need for secondary antibodies in many applications, which can reduce background and increase specificity .

What methodological considerations should be addressed when validating a new RPS4Y1 antibody?

Validation of RPS4Y1 antibodies requires rigorous testing to ensure male-specific detection without cross-reactivity. Based on published research, a comprehensive validation protocol should include:

• RT-PCR validation of gene expression:

  • Confirm RPS4Y1 mRNA expression in male samples and absence in female samples

  • Sequence PCR products to confirm specificity

• Western blotting validation:

  • Use male (e.g., HepG2) and female (e.g., HEK293) cell lysates as positive and negative controls

  • Confirm detection of a single 30 kDa band in male samples only

  • Test multiple antibody concentrations to determine optimal working dilution

• Immunoprecipitation analysis:

  • Validate antibody binding to native RPS4Y1 protein

  • Check for expected protein size (~29.4 kDa)

• Immunofluorescence studies:

  • Demonstrate specific staining in male cells

  • Confirm absence of signal in female cells

  • Include appropriate controls (primary antibody omission, isotype controls)

• Cross-reactivity testing:

  • Test against RPS4X-expressing samples to ensure no false positives

  • Evaluate potential cross-reactivity with RPS4Y2 (expressed primarily in testis)

This methodological approach ensures that newly developed antibodies specifically detect RPS4Y1 protein in male samples without false positives in female samples.

How does epitope selection impact the development of specific RPS4Y1 antibodies?

Epitope selection is crucial for developing specific RPS4Y1 antibodies due to the high homology with RPS4X. Research has shown that not all regions of the RPS4Y1 protein are equally effective as immunogens:

• Optimal epitope regions:

  • The region spanning amino acids 155-177 has been identified as particularly immunogenic and suitable for producing specific antibodies

  • This region overlaps with a stretch (155-174 aa) previously used successfully for polyclonal antibody production

• Epitope accessibility considerations:

  • RPS4Y1 is located on the exterior (cytoplasmic) side of the 40S ribosomal subunit, making certain regions more accessible

  • Accessible epitopes improve antibody binding to the native protein

• Conjugation strategies:

  • For small peptide antigens, conjugation to carrier proteins (e.g., keyhole limpet hemocyanin, KLH) improves immunogenicity

  • Pooling multiple peptide antigens can increase the chances of successful antibody production

• Epitope mapping:

  • Alignment of RPS4X and RPS4Y1 amino acid sequences identified 19 amino acid differences that can be targeted for specific antibody production

  • Focusing on regions with multiple amino acid differences increases specificity

The research demonstrates that careful epitope selection focusing on regions with the highest concentration of RPS4Y1-specific amino acids is essential for developing antibodies with the required specificity.

What is the current evidence for RPS4Y1 expression patterns across different male tissues?

Understanding RPS4Y1 expression patterns is crucial for determining appropriate experimental applications. Current evidence indicates:

This expression profile confirms that RPS4Y1 is widely expressed across male tissues, making RPS4Y1 antibodies suitable for various applications including:

• Prenatal diagnosis through detection of fetal cells in maternal circulation

• Male cell identification in mixed cell populations

• Gender determination at the cellular level

The early expression during embryonic development (E3 stage) makes RPS4Y1 particularly valuable for early fetal gender determination applications.

What are the optimal storage and handling conditions for maintaining RPS4Y1 antibody, HRP conjugated activity?

Proper storage and handling of RPS4Y1 antibody, HRP conjugated, is critical for maintaining its activity and specificity:

Storage conditions:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles that can degrade both the antibody and the HRP enzyme

• For working aliquots, store at 4°C for short-term use (1-2 weeks)

Buffer composition:

• Optimal storage buffer typically contains 50% Glycerol, 0.01M PBS, pH 7.4

• Preservatives such as 0.03% Proclin 300 help maintain stability

Handling precautions:

• HRP is sensitive to sodium azide; avoid using buffers containing this preservative

• Protect from prolonged exposure to light

• Maintain sterile conditions to prevent microbial contamination

Reconstitution (if lyophilized):

• Follow manufacturer's instructions for reconstitution

• Use sterile, purified water or recommended buffer

• Allow complete dissolution before use

Working dilution preparation:

• Prepare fresh working dilutions on the day of the experiment

• Return stock solution to -20°C/-80°C immediately after use

Following these storage and handling recommendations will help maintain antibody activity and ensure consistent experimental results.

What controls should be included when using RPS4Y1 antibody, HRP conjugated in research applications?

Inclusion of appropriate controls is essential for reliable interpretation of results when using RPS4Y1 antibody, HRP conjugated:

Essential controls for all applications:

• Positive control:

  • Male cell lysate (e.g., HepG2)

  • Male tissue sections

  • Recombinant RPS4Y1 protein

• Negative control:

  • Female cell lysate (e.g., HEK293)

  • Female tissue sections

• Technical controls:

  • Primary antibody omission control

  • Isotype control (irrelevant antibody of same isotype and host species)

  • Blocking peptide control (pre-incubation with immunogen peptide)

Application-specific controls:

Validation controls:

• Compare results from multiple antibody clones or lots

• Correlation with mRNA expression data (RT-PCR)

• Knockdown validation (if possible in male cell lines)

Including these comprehensive controls ensures that the observed signals are specific to RPS4Y1 protein and not due to non-specific binding or technical artifacts.

How can researchers optimize ELISA protocols when using RPS4Y1 antibody, HRP conjugated?

Optimizing ELISA protocols for RPS4Y1 antibody, HRP conjugated, involves several critical considerations:

Protocol optimization steps:

• Coating conditions:

  • If detecting RPS4Y1 in samples: coat with capture antibody (non-HRP conjugated anti-RPS4Y1)

  • If measuring antibody reactivity: coat with recombinant RPS4Y1 protein

  • Optimal coating buffer: typically carbonate/bicarbonate buffer (pH 9.6)

  • Coating concentration: 1-10 μg/mL

  • Coating time: 16-18 hours at 4°C

• Blocking conditions:

  • Buffer: 1-5% BSA or non-fat dry milk in PBS or TBS

  • Time: 1-2 hours at room temperature

  • Include 0.05% Tween-20 to reduce background

• Sample preparation:

  • Male cell/tissue lysates should be prepared in compatible lysis buffers

  • Dilution series to determine optimal concentration

  • Include female samples as negative controls

• Antibody dilution:

  • Initial testing range: 1:500 to 1:5000

  • Optimize based on signal-to-noise ratio

  • Dilute in blocking buffer with 0.05% Tween-20

• Detection optimization:

  • For HRP-conjugated antibodies: TMB, OPD, or enhanced chemiluminescent substrates

  • Incubation time: typically 5-30 minutes depending on substrate

  • Stop reaction when positive controls show adequate color development

• Data analysis:

  • Include standard curve if quantitation is needed

  • Calculate signal-to-background ratios

  • Set threshold based on negative controls

This optimized protocol ensures sensitive and specific detection of RPS4Y1 protein, which has been successfully used in various research applications including gender determination studies .

How does RPS4Y1 antibody compare with other Y-chromosome specific markers for male cell detection?

RPS4Y1 offers several advantages and some limitations compared to other Y-chromosome specific markers:

Key advantages of RPS4Y1:

• Expression immediately after embryonic genome activation, making it suitable for early detection

• Protein-level detection allows cellular visualization rather than just genetic detection

• Ribosomal location makes it accessible for antibody binding

• Demonstrated utility in distinguishing male from female cells in various applications

Research applications where RPS4Y1 is particularly valuable:

• Non-invasive prenatal diagnosis of X-linked diseases such as hemophilia

• Detection of rare male fetal cells in maternal blood

• Gender determination at the single-cell level

• Forensic applications requiring gender determination

The combination of early expression, widespread tissue distribution, and availability of specific antibodies makes RPS4Y1 a valuable marker for male cell detection in research settings.

What protocols have been validated for using RPS4Y1 antibody, HRP conjugated in Western blotting applications?

Western blotting with RPS4Y1 antibody, HRP conjugated, has been validated using the following protocol based on published research:

Sample preparation:

• Prepare total cell lysates from male (e.g., HepG2) and female (e.g., HEK293) cells using standard lysis buffers containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare samples containing 20-50 μg total protein with reducing loading buffer

• Heat samples at 95°C for 5 minutes

Gel electrophoresis and transfer:

• Separate proteins on SDS-PAGE (10-12% polyacrylamide gels)

• Transfer to PVDF or nitrocellulose membranes at 100V for 1 hour or 30V overnight

Immunoblotting:

• Block membranes with 5% non-fat dry milk in TBST for 1 hour at room temperature

• Incubate with RPS4Y1 antibody, HRP conjugated (1:1000 to 1:5000 dilution) in blocking buffer for 1-2 hours at room temperature or overnight at 4°C

• Wash membranes 3-5 times with TBST, 5 minutes each

• For HRP-conjugated primary antibodies, no secondary antibody is needed

• Develop using ECL substrate and detect signal using appropriate imaging system

Expected results:

• A single band of approximately 29.4-30 kDa should be detected in male samples only

• No bands should be visible in female samples

• Loading controls (β-actin, GAPDH) should show consistent bands across all samples

Troubleshooting considerations:

• If background is high, increase washing time/frequency or dilute antibody further

• If no signal is detected, reduce antibody dilution or increase protein loading

• Cross-reactivity with RPS4X would appear as bands in both male and female samples

This protocol has successfully demonstrated the specificity of RPS4Y1 antibodies in distinguishing male from female samples in research applications .

How can researchers implement RPS4Y1 antibody in immunofluorescence studies for male cell detection?

Immunofluorescence with RPS4Y1 antibody allows visual identification of male cells. For HRP-conjugated variants, either direct enzymatic detection or a two-step approach using anti-HRP secondary antibodies is possible:

Cell preparation protocol:

• Grow cells on chamber slides or coverslips

• Fix with 4% paraformaldehyde (10-15 minutes, room temperature)

• Permeabilize with 0.1-0.5% Triton X-100 (5-10 minutes)

• Block with 1-5% BSA or normal serum in PBS (1 hour)

For direct enzymatic detection:

• Incubate with diluted RPS4Y1 antibody, HRP conjugated (1:100 to 1:500) in blocking buffer (1-2 hours at room temperature or overnight at 4°C)

• Wash 3-5 times with PBS, 5 minutes each

• Incubate with tyramide-fluorophore substrate for HRP (e.g., TSA™ system) for signal amplification

• Counterstain nuclei with DAPI

• Mount and visualize using fluorescence microscopy

For fluorescent secondary detection:

• If using HRP-conjugated RPS4Y1 antibody primarily for other applications but want fluorescent detection:

  • Incubate with anti-HRP antibody conjugated to a fluorophore

  • Alternatively, use non-HRP conjugated RPS4Y1 antibody with appropriate fluorophore-conjugated secondary antibody

Expected results:

• Male cells should show positive staining for RPS4Y1

• Female cells should show no specific staining

• RPS4Y1 typically shows cytoplasmic localization due to its ribosomal association

Advanced applications:

• Dual staining with cell type-specific markers for identifying male cells in mixed populations

• Flow cytometry for quantitative analysis of male cells

• Isolation of rare male fetal cells from maternal blood for prenatal diagnosis

This immunofluorescence protocol provides a powerful tool for visual identification of male cells in research applications, particularly for non-invasive prenatal diagnosis of X-linked disorders .

What are potential sources of false positives/negatives when using RPS4Y1 antibody, and how can they be mitigated?

Understanding potential sources of error when using RPS4Y1 antibody is crucial for accurate data interpretation:

Sources of false positives:

Sources of false negatives:

Validation approaches:

• Always include male and female controls to establish baseline reactivity

• Confirm results using alternative detection methods (e.g., RT-PCR for RPS4Y1 transcript)

• Test multiple antibody concentrations to determine optimal working range

• Perform blocking peptide controls to confirm specificity

By understanding and addressing these potential sources of error, researchers can ensure reliable and reproducible results when using RPS4Y1 antibody for gender determination and other applications.

How can immunoprecipitation with RPS4Y1 antibody be optimized for studying protein interactions?

Immunoprecipitation (IP) with RPS4Y1 antibody provides insights into protein interactions and has been validated in research settings:

Optimized IP protocol based on published research:

• Cell lysis optimization:

  • Use mild lysis buffers (e.g., NP-40 buffer) to preserve native protein interactions

  • Typical composition: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, protease inhibitors

  • Incubate 30 minutes on ice with periodic vortexing

• Pre-clearing step:

  • Incubate lysate with protein G beads alone (1 hour, 4°C)

  • Remove beads by centrifugation to reduce non-specific binding

• Antibody binding:

  • Incubate pre-cleared lysate with RPS4Y1 antibody (2-5 μg) overnight at 4°C with gentle rotation

  • For HRP-conjugated antibodies, consider whether the conjugation affects binding efficiency

• Immunoprecipitation:

  • Add protein G magnetic beads and incubate 1-2 hours at 4°C

  • Collect beads using a magnetic stand

  • Wash 3-5 times with lysis buffer containing reduced detergent

• Elution and analysis:

  • Elute bound proteins with SDS sample buffer at 95°C for 5 minutes

  • Analyze by SDS-PAGE and Western blotting

Expected results and interpretation:

• IP samples should show a band at approximately 29.4 kDa corresponding to RPS4Y1 protein

• Also visible are the heavy (~50 kDa) and light (~25 kDa) chains of the antibody

• Co-precipitating proteins may include other ribosomal components or interacting partners

Validation controls:

• Use female cell lysates as negative controls

• Include isotype control antibodies

• Verify results with mass spectrometry analysis of immunoprecipitated complexes

This optimized protocol has successfully demonstrated the ability of RPS4Y1 antibodies to recognize the native protein, enabling studies of protein-protein interactions in male cells .

What troubleshooting approaches are recommended for common issues with RPS4Y1 antibody, HRP conjugated?

When encountering issues with RPS4Y1 antibody, HRP conjugated, the following troubleshooting approaches are recommended:

Application-specific troubleshooting:

For ELISA:

• Optimize coating conditions and antibody concentrations

• Consider different blocking agents (BSA, casein, normal serum)

• Test various substrate incubation times

For Immunofluorescence:

• Optimize fixation and permeabilization conditions

• Consider signal amplification methods for low abundance detection

• Use confocal microscopy for improved signal localization

For Immunoprecipitation:

• Increase antibody amount or incubation time

• Test different lysis buffer compositions

• Consider crosslinking antibody to beads to reduce IgG contamination

These troubleshooting approaches address common issues encountered when working with RPS4Y1 antibody, HRP conjugated, across various research applications.