RTF1 Antibody, HRP conjugated

What is the RTF1 protein and why is it important in research?

RTF1 (RNA polymerase-associated protein RTF1 homolog) is a key component of the PAF1 complex (PAF1C) which performs multiple functions during transcription by RNA polymerase II. This protein is critically involved in transcriptional elongation, acting both independently and synergistically with TCEA1 and in cooperation with the DSIF complex and HTATSF1 . RTF1 plays essential roles in the regulation of development and maintenance of embryonic stem cell pluripotency. The protein is required for transcription of Hox and Wnt target genes, which are fundamental in developmental biology research .

Additionally, RTF1 participates in critical cellular processes including histone modifications such as ubiquitination of histone H2B and methylation on histone H3 'Lys-4' (H3K4me3). It also contributes to hematopoiesis and stimulates transcriptional activity of KMT2A/MLL1 . Given its involvement in these fundamental biological processes, RTF1 has become an important target for researchers studying transcriptional regulation, chromatin remodeling, and developmental biology.

What applications are HRP-conjugated RTF1 antibodies most suitable for?

HRP (Horseradish Peroxidase)-conjugated RTF1 antibodies are particularly well-suited for enzyme-linked immunosorbent assays (ELISA) and other applications requiring sensitive detection with enzymatic amplification . The HRP conjugation enables colorimetric, chemiluminescent, or fluorescent detection when appropriate substrates are used, making these antibodies highly versatile for quantitative and qualitative analyses.

For ELISA applications, HRP-conjugated RTF1 antibodies provide excellent sensitivity and a broad dynamic range of detection . They are also useful in immunohistochemistry applications where signal amplification is required, although unconjugated primary antibodies followed by HRP-conjugated secondary antibodies are more commonly used for this purpose. The specificity of the antibody combined with the enzymatic amplification provided by HRP makes these conjugates particularly valuable in experiments where target protein expression is low or sample quantity is limited.

How do you validate the specificity of an RTF1 antibody before experimental use?

Validating antibody specificity is a critical step before initiating experiments with RTF1 antibodies. A comprehensive validation approach should include multiple methodologies. Western blotting is a primary validation method where the antibody should detect a band at the predicted molecular weight of RTF1 (approximately 80 kDa) . The appearance of a single, clean band at the expected molecular weight provides good evidence of specificity.

Additional validation can be performed through immunoprecipitation (IP) followed by mass spectrometry to confirm that the antibody is pulling down authentic RTF1 protein . For immunohistochemistry or immunofluorescence applications, comparing staining patterns with literature reports and using positive and negative control tissues is essential. Testing the antibody in cells or tissues where RTF1 has been knocked down or knocked out via siRNA or CRISPR-Cas9 technologies provides compelling evidence of specificity. Lastly, comparing results from multiple antibodies targeting different epitopes of RTF1 can provide confirmatory evidence of specificity when staining patterns align .

What is the optimal protocol for using HRP-conjugated RTF1 antibodies in ELISA?

The optimal protocol for ELISA using HRP-conjugated RTF1 antibodies requires careful optimization of several parameters. Begin by coating high-binding ELISA plates with your capture antibody or antigen at a concentration of 1-5 μg/ml in carbonate buffer (pH 9.6) overnight at 4°C. After washing with PBS-T (PBS containing 0.05% Tween-20), block non-specific binding sites with 1-3% BSA or 5% non-fat dry milk in PBS for 1-2 hours at room temperature .

For direct ELISA, add your samples containing RTF1 protein in appropriate dilutions. For sandwich ELISA, first add samples, followed by washing and then addition of the HRP-conjugated RTF1 antibody. The HRP-conjugated RTF1 antibody should be diluted according to manufacturer recommendations, typically in the range of 1:1000 to 1:5000 in blocking buffer . Incubate for 1-2 hours at room temperature or overnight at 4°C. Following thorough washing (4-6 times with PBS-T), add appropriate HRP substrate (TMB, ABTS, or chemiluminescent substrates). Develop the reaction and measure absorbance or luminescence using a plate reader. Include proper negative controls and standard curves to ensure accurate quantification and specificity .

How should RTF1 antibodies be stored to maintain optimal activity?

Proper storage of RTF1 antibodies, especially HRP-conjugated versions, is crucial to maintain optimal activity and extend shelf life. Generally, RTF1 antibodies should be stored at 4°C and should not be frozen to avoid freeze-thaw cycles that can degrade antibody quality . Aliquoting the antibody upon receipt can help minimize freeze-thaw cycles if long-term storage is necessary.

What is the recommended dilution range for RTF1 antibodies in Western blotting?

The recommended dilution range for RTF1 antibodies in Western blotting varies depending on the specific antibody formulation, but typical working dilutions range from 1:1000 to 1:10,000 . For instance, the RTF1 antibody ab184570 is recommended to be used at a 1:1000 dilution for optimal results in Western blotting . Polyclonal RTF1 antibodies like NB100-61052 may be used at dilutions ranging from 1:2000 to 1:10,000 .

The optimal dilution should be determined empirically for each experimental setup and antibody lot. Start with the manufacturer's recommended dilution and adjust as needed based on signal intensity and background levels. When working with HRP-conjugated RTF1 antibodies directly, the dilutions might need to be adjusted compared to unconjugated primary antibodies. For Western blotting, a blocking buffer containing 3-5% non-fat dry milk or BSA in TBS-T is typically recommended to minimize background . Incubation times can range from 1 hour at room temperature to overnight at 4°C, depending on antibody affinity and target abundance. Always include appropriate positive and negative controls to validate the specificity of the signal and optimize dilution factors.

How can RTF1 antibodies be used to study chromatin modifications and transcriptional regulation?

RTF1 antibodies are powerful tools for investigating chromatin modifications and transcriptional regulation due to RTF1's critical role in these processes. Chromatin immunoprecipitation (ChIP) assays using RTF1 antibodies allow researchers to map RTF1 binding sites genome-wide and correlate these with specific histone modifications. RTF1, as part of the PAF1 complex, is involved in histone H2B ubiquitination and H3K4 methylation, which are key markers of active transcription .

For advanced applications, RTF1 antibodies can be utilized in ChIP-seq experiments to identify genome-wide binding profiles of RTF1 and correlate these with specific gene expression patterns. Co-immunoprecipitation (Co-IP) using RTF1 antibodies helps identify protein-protein interactions within the transcriptional machinery, providing insights into the composition and dynamics of RTF1-containing complexes. When combined with sequential ChIP (ChIP-reChIP), researchers can determine co-occupancy of RTF1 with other transcription factors or modified histones at specific genomic loci .

Additionally, proximity ligation assays (PLA) using RTF1 antibodies enable visualization of protein interactions in situ, helping to understand the spatial organization of transcriptional complexes within the nucleus. For studies focusing on the kinetics of transcriptional regulation, RTF1 antibodies can be employed in conjunction with techniques like fluorescence recovery after photobleaching (FRAP) or live-cell imaging using fluorescently tagged secondary antibodies to monitor dynamic associations of RTF1 with chromatin during transcription elongation .

What are the considerations when using RTF1 antibodies in multiplex immunofluorescence studies?

Multiplex immunofluorescence studies involving RTF1 antibodies require careful consideration of several factors to ensure successful results. Primary among these is antibody compatibility—RTF1 antibodies must be selected to minimize cross-reactivity with other primary antibodies in the multiplex panel. Ideally, primary antibodies should be from different host species or utilize isotype-specific secondary antibodies to prevent cross-reactivity .

Epitope accessibility is another critical consideration. RTF1 localizes primarily to the nucleus, requiring appropriate fixation and permeabilization protocols to ensure antibody penetration without destroying antigenic epitopes. Paraformaldehyde fixation (4%) followed by Triton X-100 permeabilization often works well for nuclear proteins like RTF1 . The order of antibody application can significantly impact results, particularly when using signal amplification systems. Generally, lower abundance targets like RTF1 should be detected first with amplification steps if needed.

For spectral overlap considerations, select fluorophores with minimal spectral overlap for different targets. When using HRP-conjugated RTF1 antibodies in multiplex studies, employ sequential tyramide signal amplification (TSA) with microwave treatment or chemical stripping between rounds to prevent cross-talk. Additionally, include proper controls for each antibody individually to establish specificity before combining them in multiplex format. Single-stain controls are essential for spectral unmixing in multispectral imaging systems, and fluorescence minus one (FMO) controls help establish gating boundaries in flow cytometry applications .

What approaches can resolve contradictory results when using different RTF1 antibodies?

When faced with contradictory results using different RTF1 antibodies, a systematic troubleshooting approach is necessary. Begin by carefully examining the epitope locations of each antibody. Different RTF1 antibodies may target distinct regions of the protein (N-terminal, middle region, or C-terminal), which could be differentially accessible depending on protein conformation, interactions, or post-translational modifications . For instance, one antibody targets RTF1 at amino acids 441-470 in the middle region , while others target the C-terminus or different regions.

Validation through orthogonal techniques is essential for resolving discrepancies. Compare results from multiple methods such as Western blotting, immunoprecipitation, immunofluorescence, and functional assays. Knockdown or knockout controls using siRNA or CRISPR-Cas9 targeting RTF1 can definitively establish antibody specificity—a genuine RTF1 antibody should show reduced or absent signal in these samples .

Consider the possibility of detecting different isoforms or post-translationally modified forms of RTF1. Mass spectrometry analysis of immunoprecipitated proteins can help identify exactly what each antibody is detecting. Additionally, examine antibody performance across different experimental conditions such as fixation methods, blocking reagents, and antigen retrieval protocols, as these can significantly affect epitope accessibility and antibody binding . Finally, consult literature and collaborators who have experience with these specific antibodies, and consider reaching out to the manufacturers for technical support and advice on resolving discrepancies.

How can I optimize antigen retrieval for RTF1 detection in formalin-fixed paraffin-embedded tissues?

Optimizing antigen retrieval for RTF1 detection in FFPE tissues is critical for successful immunohistochemistry experiments. Heat-induced epitope retrieval (HIER) with EDTA buffer at pH 9.0 is generally recommended for RTF1 detection, as noted in several antibody protocols . This alkaline buffer helps break protein cross-links formed during formalin fixation, making the RTF1 epitopes more accessible to antibodies.

The optimal procedure involves deparaffinizing sections completely in xylene followed by rehydration through an ethanol gradient. For HIER, immerse slides in EDTA buffer (pH 9.0) and heat in a pressure cooker or microwave for 15-20 minutes, followed by cooling to room temperature for 20 minutes. This method has been demonstrated to effectively retrieve RTF1 antigens in various tissue types, including stomach, thyroid, and skin samples . Alternatively, some protocols recommend citrate buffer (pH 6.0) for RTF1 retrieval in certain tissue types .

If background staining remains problematic after standard retrieval, consider additional blocking steps with 3% hydrogen peroxide to quench endogenous peroxidase activity, especially when using HRP-conjugated detection systems. Block endogenous biotin if using biotin-based detection methods. For tissues with high background, extend the protein blocking step using 5-10% normal serum from the same species as the secondary antibody. Systematic optimization by testing multiple retrieval conditions (buffer composition, pH, heating time, and cooling method) may be necessary to determine the ideal protocol for specific tissue types and fixation conditions .

What controls are essential when using RTF1 antibodies in research applications?

Implementing appropriate controls when using RTF1 antibodies is fundamental to experimental rigor and result interpretation. Positive tissue or cell controls known to express RTF1 should be included in every experiment. Human and mouse tissues such as thyroid, stomach, and skin have been documented to express detectable levels of RTF1 and serve as excellent positive controls .

Negative controls are equally important and should include isotype controls (using non-specific antibodies of the same isotype and concentration as the RTF1 antibody) to identify any non-specific binding. For tissues or cells where RTF1 expression is absent or minimized through genetic manipulation (siRNA knockdown or CRISPR knockout), these serve as biological negative controls that authenticate antibody specificity .

For HRP-conjugated RTF1 antibodies specifically, additional controls should include substrate-only controls to identify any non-enzymatic reaction with the substrate and endogenous enzyme controls to detect endogenous peroxidase activity. When performing multiplex experiments, single-stain controls for each antibody are essential to establish proper signal separation. For quantitative applications like ELISA or Western blotting, standard curves using recombinant RTF1 protein at known concentrations should be included to ensure accurate quantification. Finally, technical replicate controls across multiple experiments help establish reproducibility and reliability of the observed results .

How can I troubleshoot weak or absent signal when using RTF1 antibodies?

Troubleshooting weak or absent signals when using RTF1 antibodies requires a systematic approach examining multiple aspects of the experimental procedure. Begin by verifying RTF1 expression in your experimental system, as low or absent expression of the target protein is a common cause of weak signals. Consult literature or databases for expected expression levels in your specific cell type or tissue .

For fixation-related issues, optimize your fixation protocol, as excessive fixation can mask epitopes while insufficient fixation may result in poor tissue preservation. For FFPE samples, ensure complete deparaffinization and optimize antigen retrieval as described previously. Test different retrieval methods, including EDTA buffer pH 9.0 and citrate buffer pH 6.0 .

Antibody-related factors may also contribute to signal issues. Check antibody quality and storage conditions, as improper storage can lead to antibody degradation. Try titrating the antibody concentration—both too high and too low concentrations can result in suboptimal results. Consider testing different antibody clones or lots, as epitope recognition can vary between antibody preparations . For HRP-conjugated antibodies specifically, verify that the HRP enzyme is still active by testing the antibody with a known positive sample or control system.

Detection system optimization is another crucial element. For HRP-conjugated antibodies, ensure your substrate is fresh and properly prepared. Consider using signal amplification methods such as tyramide signal amplification (TSA) for very low abundance targets. Increase incubation times for primary antibody (overnight at 4°C instead of 1 hour at room temperature) to allow more time for antibody-antigen binding . Finally, reduce washing stringency if you suspect you're washing away specific signal, or switch to a more sensitive detection method (e.g., from colorimetric to chemiluminescent for Western blots or immunohistochemistry).

What is the expression profile of RTF1 across different human tissues and cell lines?

RTF1 expression demonstrates distinctive patterns across human tissues and cell lines, reflecting its fundamental role in transcriptional regulation. Based on immunohistochemistry studies using validated RTF1 antibodies, moderate to strong nuclear expression is observed in epithelial cells of various tissues, consistent with RTF1's function in transcriptional processes .

In cell lines, RTF1 expression is particularly abundant in rapidly proliferating cells. HeLa cells show strong nuclear localization of RTF1 as demonstrated by immunofluorescence studies . Several cancer cell lines exhibit elevated RTF1 expression compared to their normal tissue counterparts, suggesting a potential role in cancer biology. Expression levels also appear to correlate with proliferation status, with higher expression in actively dividing cells compared to quiescent populations .

How does RTF1 interact with other components of the PAF1 complex in transcriptional regulation?

RTF1 functions as a critical component of the PAF1 complex (PAF1C), engaging in multiple protein-protein interactions that collectively regulate transcriptional processes. Immunoprecipitation and mass spectrometry studies have revealed that RTF1 directly interacts with other core PAF1C components including PAF1, CTR9, CDC73, and LEO1 to form a stable complex .

RTF1's histone modification domain (HMD) is specifically required for histone H2B ubiquitination and subsequent H3K4 methylation. Through its Plus3 domain, RTF1 directly binds to single-stranded DNA, potentially stabilizing R-loops or other nucleic acid structures during transcription . The protein also contains a C-terminal region that interacts with the cleavage and polyadenylation machinery, linking transcription elongation to mRNA 3' end formation.

During heat shock or other stress responses, RTF1 demonstrates altered interaction patterns, with increased association with heat shock transcription factors to facilitate rapid induction of stress-responsive genes . In embryonic stem cells, RTF1 shows enhanced interaction with the SET1 complex in a CXXC1-dependent manner, specifically on genes involved in maintaining pluripotency. This specialized interaction pattern appears critical for the trimethylation of H3K4 on development-related genes .

What is the role of RTF1 in embryonic development and stem cell maintenance?

RTF1 plays crucial roles in embryonic development and stem cell maintenance through its functions in regulating gene expression patterns essential for proper developmental progression. Studies using RTF1 antibodies in developmental systems have revealed that RTF1 is required for the transcription of Hox and Wnt target genes, which are master regulators of body plan establishment and tissue patterning during embryogenesis .

In embryonic stem cells, RTF1 contributes to the maintenance of pluripotency by facilitating the trimethylation of histone H3 'Lys-4' (H3K4me3) on genes involved in stem cell self-renewal and pluripotency. This function is synergistic with CXXC1, suggesting involvement of a SET1 complex in establishing proper chromatin states necessary for pluripotency . Immunofluorescence and ChIP-seq studies have demonstrated RTF1 occupancy at promoters and gene bodies of key pluripotency factors in embryonic stem cells.