GTF2F1 Antibody, HRP conjugated

What is GTF2F1 and what is its biological function?

GTF2F1, also known as RAP74, is a general transcription factor that serves as the 74 kDa subunit of transcription factor IIF (TFIIF). It plays a crucial role in the transcription machinery by binding to RNA polymerase II and facilitating its recruitment to the initiation complex in collaboration with TFIIB . ChIP-seq studies have revealed that GTF2F1 exhibits a distinctive binding pattern centered on transcription start sites (TSS), reflecting its fundamental role in transcription initiation .

Additionally, GTF2F1 promotes transcription elongation and is subject to post-translational modifications, including phosphorylation by TAF1 on serine residues . This phosphorylation may serve as a regulatory mechanism for its activity in the transcription process. The protein's central role in the core transcriptional machinery makes it an important subject for research into gene expression regulation.

What characteristics define HRP-conjugated GTF2F1 antibodies?

HRP-conjugated GTF2F1 antibodies are immunological reagents where horseradish peroxidase enzyme is directly linked to antibodies targeting the GTF2F1 protein. These conjugated antibodies offer several technical advantages:

• Direct detection without secondary antibodies, simplifying experimental workflows

• Reduced background signal in certain applications

• Compatibility with chromogenic and chemiluminescent detection methods

• Enhanced sensitivity for detecting low-abundance transcription factors

Typically, these antibodies are produced by immunizing rabbits with recombinant human GTF2F1 protein (specifically amino acids 1-249) . The purified antibodies are then conjugated to HRP enzyme while maintaining their specificity for the target protein. The resulting reagent enables researchers to directly visualize GTF2F1 in various experimental applications.

What experimental applications utilize GTF2F1 antibodies?

GTF2F1 antibodies find utility in multiple research applications investigating transcriptional regulation:

HRP-conjugated versions are particularly valuable for ELISA applications, where direct enzyme linkage provides cleaner signal detection. For Western blotting, these antibodies allow for one-step detection protocols, eliminating the need for separate secondary antibody incubation steps . In chromatin immunoprecipitation experiments, GTF2F1 antibodies have been crucial for mapping the genome-wide distribution of this factor and understanding its association with active gene promoters .

Why does the observed molecular weight of GTF2F1 differ from calculations?

The calculated molecular weight of GTF2F1 is approximately 58 kDa, but Western blotting typically reveals a band at approximately 74 kDa . This discrepancy represents a common phenomenon in protein analysis that can be attributed to several factors:

• Post-translational modifications: GTF2F1 undergoes phosphorylation by TAF1 , which can significantly increase its apparent molecular weight

• Protein structural properties: The three-dimensional conformation of GTF2F1 may affect its migration rate in gel electrophoresis

• Amino acid composition: Certain amino acid compositions can cause anomalous migration during SDS-PAGE

• Protein domain architecture: Specific structural domains may interact differently with SDS, affecting mobility

As noted in technical documentation: "Western blotting is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. Different proteins can be divided into bands based on different mobility rates. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

When multiple bands are observed, this may indicate the presence of different modified forms of GTF2F1 in the sample simultaneously .

What are the optimal conditions for Western blotting with GTF2F1 antibodies?

Achieving robust and specific detection of GTF2F1 by Western blotting requires careful optimization of several parameters:

Sample Preparation:

• Include phosphatase inhibitors in lysis buffers to preserve phosphorylated forms of GTF2F1

• Ensure complete nuclear protein extraction, as GTF2F1 is predominantly nuclear

• Denature samples thoroughly in loading buffer containing SDS and reducing agents

Gel Electrophoresis:

• Use 8-10% polyacrylamide gels to properly resolve the 74 kDa GTF2F1 band

• Include molecular weight markers that span the 50-100 kDa range

• Load positive control samples from validated cell lines (HeLa, K-562, Jurkat)

Transfer and Detection:

• For unconjugated antibodies, use dilutions of 1:500-1:2000

• For HRP-conjugated antibodies, optimize starting from manufacturer recommendations

• Block membranes with 5% non-fat milk or BSA in TBS-T

• Incubate with primary antibody overnight at 4°C for optimal specificity

• Wash thoroughly to minimize background signal

Controls and Validation:

• Include positive control lysates from validated cell lines

• Consider running lysates from cells with GTF2F1 knockdown as negative controls

• Be aware that the observed molecular weight (74 kDa) differs from the calculated value (58 kDa)

The discrepancy between calculated and observed molecular weights should be anticipated and does not necessarily indicate lack of specificity, as this is a documented characteristic of GTF2F1 detection .

How should immunohistochemistry be optimized for GTF2F1 detection?

For successful immunohistochemical detection of GTF2F1 in tissue samples, consider these methodological recommendations:

Tissue Preparation:

• Formalin-fixed, paraffin-embedded (FFPE) tissue sections work well for GTF2F1 detection

• Section thickness of 4-6 μm provides optimal results

• Mount sections on positively-charged slides to prevent tissue loss

Antigen Retrieval:

• Heat-induced epitope retrieval is essential for exposing GTF2F1 epitopes

• Use TE buffer at pH 9.0 as the primary recommendation

• Alternatively, citrate buffer at pH 6.0 may be effective for some tissue types

• Heating duration and temperature should be optimized for each tissue type

Antibody Application:

• Dilute GTF2F1 antibodies at 1:50-1:200 for IHC applications

• For HRP-conjugated versions, follow manufacturer's specific recommendations

• Incubate primary antibody overnight at 4°C or for 1-2 hours at room temperature

• Include both positive and negative control tissues in each staining run

Detection and Visualization:

• For unconjugated antibodies, use appropriate detection systems based on host species

• For HRP-conjugated antibodies, proceed directly to chromogen development

• DAB (3,3'-diaminobenzidine) provides a stable brown reaction product

• Counterstain with hematoxylin for nuclear visualization

Human colon carcinoma and esophagus tissues have been verified for positive GTF2F1 staining and serve as excellent positive controls .

What controls should be included in GTF2F1 antibody experiments?

Rigorous experimental design requires appropriate controls to validate specificity and ensure reproducible results:

Positive Controls:

• Cell lysates: HEK-293T, HeLa, Jurkat, and K-562 cells have been verified for GTF2F1 expression

• Tissue sections: Human colon carcinoma and esophagus samples show reliable GTF2F1 staining

• Recombinant protein: Purified GTF2F1 can serve as a positive control for Western blotting

Negative Controls:

• Primary antibody omission: Replace GTF2F1 antibody with antibody diluent alone

• Isotype control: Use non-specific rabbit IgG at the same concentration

• Absorption control: Pre-incubate antibody with immunizing peptide to block specific binding

• RNAi samples: Lysates from cells treated with GTF2F1-targeting siRNA should show reduced signal

Methodological Controls:

• Molecular weight verification: Confirm band appears at expected size (~74 kDa)

• Loading controls: Include housekeeping proteins (GAPDH, β-actin) for Western blotting

• Multiple antibody approach: Use antibodies targeting different GTF2F1 epitopes

• Cross-application validation: Confirm findings using multiple detection methods

Proper documentation of all controls is essential for publication and reproducibility, including detailed antibody information (source, catalog number, lot) and experimental conditions.

What storage and handling practices maximize GTF2F1 antibody performance?

Proper storage and handling are critical for maintaining antibody functionality and experimental reproducibility:

Storage Conditions:

• Store unconjugated GTF2F1 antibodies at -20°C

• HRP-conjugated antibodies may have specific storage requirements (typically -20°C)

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Consider aliquoting stock solutions to minimize freeze-thaw events

Buffer Composition:

• Most GTF2F1 antibodies are supplied in phosphate buffered solution at pH 7.4

• Typical formulations contain 0.05% stabilizer and 50% glycerol

• HRP-conjugated versions may contain preservatives such as 0.03% Proclin 300

• Do not add sodium azide to HRP-conjugated antibodies as it inhibits peroxidase activity

Working Solution Handling:

• Thaw antibodies completely before use

• Mix gently by inversion or gentle flicking (avoid vortexing)

• Keep on ice during experimental procedures

• Return to -20°C promptly after use

Stability Considerations:

• Typical shelf life is 12 months when stored properly

• Document first use date and monitor performance over time

• HRP-conjugated antibodies may have shorter shelf life due to enzyme stability

• Avoid exposure to direct light, particularly for conjugated antibodies

For shipping and receipt: "The product is shipped with ice pack, upon receipt, store it immediately at the temperature recommended."

How can GTF2F1 antibodies elucidate transcription initiation mechanisms?

GTF2F1 antibodies offer powerful tools for investigating transcription initiation machinery through several advanced approaches:

Protein Complex Analysis:

• Co-immunoprecipitation with GTF2F1 antibodies can isolate intact transcription initiation complexes

• Mass spectrometry analysis of precipitated complexes can identify novel interaction partners

• Sequential immunoprecipitation can determine co-occupancy of GTF2F1 with other factors

Genomic Binding Studies:

• ChIP-seq reveals genome-wide GTF2F1 binding sites, typically centered on transcription start sites

• This distinctive TSS-centered binding pattern distinguishes GTF2F1 from other RNA-binding proteins that associate with regions downstream of the TSS

• The precise positioning provides insights into active promoter architecture and dynamics

Functional Assays:

• Depletion of GTF2F1 followed by transcription assays can define its requirement for specific genes

• In vitro transcription systems with purified components can determine the mechanistic contribution

• Reporter assays with mutated GTF2F1 can elucidate structure-function relationships

Dynamic Regulation Studies:

• ChIP-qPCR time course experiments can track GTF2F1 recruitment during transcriptional activation

• Phospho-specific antibodies can monitor the regulatory state of GTF2F1 during transcription

• Immunofluorescence microscopy can visualize nuclear localization and redistribution

These approaches collectively provide comprehensive insights into how GTF2F1 contributes to transcription initiation complex formation, promoter recognition, and regulatory mechanisms controlling gene expression.

What insights can ChIP-seq analysis of GTF2F1 provide?

ChIP-seq studies of GTF2F1 have revealed significant insights into transcriptional regulation across different cellular contexts:

Binding Pattern Characteristics:

• GTF2F1 exhibits a distinctive binding profile centered precisely on transcription start sites

• This pattern distinguishes it from many RNA-binding proteins that associate primarily with downstream regions

• The TSS-centered binding reflects GTF2F1's role as a core component of the transcription initiation machinery

Co-binding Relationships:
Analysis of ENCODE data across multiple cell lines reveals coordinated binding patterns:

Regulatory Network Insights:

• GTF2F1 clusters with distinct functional groups in different cell types

• In HeLa-S3, GTF2F1 associates with TBP, GTFs, MYC, MAX, MXI1, and CHD2

• GTF2F1 does not cluster with chromatin organization factors like CTCF/RAD21/SMC3

• These associations provide insights into cell type-specific transcriptional programs

Methodological Considerations:

• GTF2F1 ChIP-seq can serve as a proxy for identifying active promoters

• Integration with RNA-seq data can correlate binding with transcriptional output

• Combining with histone modification data provides chromatin context information

• Differential binding analysis across conditions can identify regulated gene sets

These genome-wide analyses continue to expand our understanding of how GTF2F1 functions within the complex regulatory networks controlling gene expression.

How does GTF2F1 interact with other transcription machinery components?

GTF2F1 participates in an intricate network of interactions within the transcriptional apparatus:

Core Transcription Factor Interactions:

• GTF2F1 functions as part of TFIIF, binding directly to RNA polymerase II

• It collaborates with TFIIB to help recruit polymerase to the initiation complex

• Forms functional associations with TBP (TATA-binding protein) and TAFs at promoters

• TAF1 phosphorylates GTF2F1 on serine residues, potentially regulating its activity

Functional Co-binding Patterns:
ENCODE ChIP-seq analyses across multiple cell lines have revealed consistent co-binding patterns with:

• Core transcriptional machinery components (TBP, GTFs)

• Cell-cycle regulators (E2Fs, MYC, MAX)

• Cell type-specific transcription factors

• Chromatin modifiers (CHD2, RCOR1)

Regulatory Hierarchies:

• Sequence-specific transcription factors may recruit or stabilize GTF2F1 binding

• GTF2F1 occupancy often correlates with binding of upstream activators

• The TSS-centered binding pattern reflects its role in transcription initiation

• This positioning distinguishes it from factors involved primarily in elongation

Structural Considerations:

• As part of TFIIF, GTF2F1 contributes to the structural organization of the preinitiation complex

• Its interaction with RNA polymerase II stabilizes the enzyme on promoter DNA

• The elongation-promoting activity suggests functional roles beyond initiation

Understanding these interaction networks is essential for deciphering the complex regulatory mechanisms governing gene expression in different cellular contexts.

What role does post-translational modification play in GTF2F1 function?

Post-translational modifications, particularly phosphorylation, critically regulate GTF2F1 activity:

Phosphorylation Mechanisms:

• TAF1, the largest component of TFIID, phosphorylates GTF2F1 on serine residues

• TAF1 contains both N- and C-terminal Ser/Thr kinase domains capable of this modification

• This phosphorylation represents a direct regulatory connection between TFIID and TFIIF

• The modification may occur within the context of the assembled preinitiation complex

Functional Significance:

• Phosphorylation may regulate GTF2F1's interaction with RNA polymerase II

• Modified GTF2F1 can exhibit altered DNA binding properties

• Phosphorylation status may influence transcription elongation rates

• These modifications potentially coordinate transcription with cell cycle progression, as TAF1 is essential for G1 phase progression

Experimental Detection:

• Phosphorylation contributes to the observed molecular weight difference between calculated (58 kDa) and detected (74 kDa) GTF2F1

• Multiple bands may appear in Western blots representing differentially phosphorylated forms

• Phosphatase treatment of samples can confirm phosphorylation status

• Phospho-specific antibodies may detect specific modified residues

This regulated phosphorylation exemplifies how post-translational modifications fine-tune the core transcriptional machinery to respond to cellular signals and developmental cues.

What causes variable GTF2F1 detection patterns across different cell types?

Variations in GTF2F1 detection across cell types stem from both biological differences and technical considerations:

Biological Factors:

• Expression level differences: GTF2F1 abundance varies between cell types

• Phosphorylation patterns: Cell-specific signaling affects post-translational modifications

• Protein complex formation: Different binding partners may mask certain epitopes

• Chromatin context: The local chromatin environment influences GTF2F1 accessibility

• Nuclear architecture: Organization of transcription factories affects distribution

Technical Variables:

• Fixation sensitivity: Different fixation methods may differentially preserve epitopes

• Antibody accessibility: Nuclear penetration can vary between cell types

• Background signal: Endogenous peroxidase activity varies across tissues

• Cross-reactivity profiles: Non-specific targets may be differentially expressed

Interpretation Strategies:

• Compare binding patterns with transcriptional activity data

• Use multiple antibodies targeting different GTF2F1 epitopes

• Include validated positive control cell types (HeLa, K-562)

• Consider cell type-specific contexts when interpreting results

Understanding these variations is crucial for accurate data interpretation and can reveal important insights about context-dependent transcriptional regulation.

How can researchers verify GTF2F1 antibody specificity?

Validating antibody specificity is essential for generating reliable research data:

Molecular Verification Approaches:

• Western blot analysis: Confirm single band at the expected molecular weight (~74 kDa)

• Immunoprecipitation followed by mass spectrometry: Identify pulled-down proteins

• RNA interference: Demonstrate reduced signal after GTF2F1 knockdown

• CRISPR/Cas9 knockout: Show complete signal loss in knockout cells

• Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

Bioinformatic Analysis:

• Epitope conservation: Verify evolutionary conservation of the target epitope

• Cross-reactivity prediction: Analyze potential binding to related proteins

• ChIP-seq peak distribution: Confirm expected TSS-centered binding pattern

Control Samples:

• Use verified positive samples: Human colon carcinoma, HeLa cells

• Compare results from different antibodies targeting GTF2F1

• Include isotype controls to establish background signal levels

Documentation Standards:

• Record complete antibody information including source, catalog number, and lot

• Document all experimental conditions used for validation

• Include images of complete Western blots with molecular weight markers

• Maintain raw data from validation experiments

These comprehensive validation approaches ensure experimental reproducibility and data reliability.

What strategies address inconsistent results with GTF2F1 antibodies?

When facing inconsistent experimental outcomes with GTF2F1 antibodies, systematic troubleshooting is essential:

Antibody-Related Variables:

• Lot-to-lot variation: Test new lots against previous ones before use in critical experiments

• Storage conditions: Ensure proper storage at -20°C and avoid freeze-thaw cycles

• Working concentration: Optimize dilutions for each application and lot

• Age of antibody: Monitor performance over time as antibodies can degrade

Sample Preparation Factors:

• Fixation parameters: Standardize fixation duration and conditions

• Protein extraction: Ensure complete nuclear protein extraction

• Phosphorylation status: Use consistent phosphatase inhibitor protocols

• Sample handling: Minimize protein degradation with proper protease inhibitors

Protocol Standardization:

• Antigen retrieval: Use consistent buffer composition (TE buffer pH 9.0 or citrate buffer pH 6.0)

• Blocking conditions: Standardize blocking reagents and duration

• Incubation parameters: Maintain consistent temperature, time, and antibody concentration

• Washing procedures: Use identical washing buffer composition and times

Documentation and Controls:

• Implement detailed protocol documentation with each experimental variable

• Include internal controls in every experiment

• Process all experimental samples simultaneously when possible

• Maintain consistent detection system parameters

Addressing these variables systematically can identify the source of inconsistency and establish reliable experimental conditions for GTF2F1 detection.

How can cross-reactivity issues be identified and mitigated?

Cross-reactivity represents a significant challenge in antibody-based research that requires systematic identification and management:

Identification Methods:

• Western blot analysis: Look for unexpected bands that may indicate cross-reactivity

• Multiple antibody comparison: Test different antibodies targeting distinct GTF2F1 epitopes

• RNAi validation: Compare signal patterns with GTF2F1-depleted samples

• Peptide competition: Pre-incubate with immunizing peptide to block specific binding

Mitigation Strategies:

• Antibody selection: Choose affinity-purified antibodies with validated specificity

• Dilution optimization: Test serial dilutions to find optimal signal-to-noise ratio

• Blocking enhancement: Optimize blocking conditions to reduce non-specific binding

• Pre-absorption: Incubate antibody with non-target tissues to deplete cross-reactive antibodies

Analytical Considerations:

• Document all observed bands and their molecular weights

• Consider that the 74 kDa observed GTF2F1 band differs from the calculated 58 kDa

• Multiple bands may represent different modified forms rather than cross-reactivity

• Compare results across different detection methods for confirmation

Application-Specific Approaches:

• For Western blotting: Include full blot images with molecular weight markers

• For IHC: Test antibody on tissues known to be negative for GTF2F1

• For ChIP: Verify expected binding pattern centered on transcription start sites

• For flow cytometry: Use isotype controls and GTF2F1-depleted cells

By systematically addressing cross-reactivity concerns, researchers can enhance the reliability and specificity of their GTF2F1 antibody-based experiments.