ZNF549 Antibody

What is ZNF549 and what cellular systems express this protein?

ZNF549 (zinc finger protein 549, also known as FLJ34917) is a human zinc finger protein with a calculated molecular weight of 74 kDa and consists of 640 amino acids . Based on current research findings, ZNF549 has been detected in multiple human cell lines including SH-SY5Y (neuroblastoma), A549 (lung carcinoma), HeLa (cervical carcinoma), and SMMC-7721 (hepatoma) cells . While the precise cellular function remains under investigation, as a zinc finger protein, it likely plays a role in transcriptional regulation by binding to specific DNA sequences. The UNIPROT ID for this protein is Q6P9A3 and its gene ID (NCBI) is 256051 .

What types of ZNF549 antibodies are available for research applications?

Current research primarily utilizes rabbit polyclonal antibodies against ZNF549. These antibodies are typically generated by immunizing rabbits with ZNF549 recombinant protein (for example, using accession number NM_001199295) . The antibodies are purified using antigen affinity methods to ensure specificity . While monoclonal antibodies might offer greater specificity in some applications, the validated polyclonal antibodies against ZNF549 have demonstrated reliable detection in multiple experimental systems across various applications.

What applications have been validated for ZNF549 antibodies?

ZNF549 antibodies have been successfully validated for multiple research applications:

The observed molecular weight in Western blot applications is consistently 74 kDa, which aligns with the theoretical molecular weight .

What are the optimal conditions for Western blot detection of ZNF549?

For optimal Western blot detection of ZNF549, researchers should consider the following protocol elements:

• Sample preparation: Standard SDS-PAGE sample preparation methods are suitable, with samples from SH-SY5Y, A549, HeLa, and SMMC-7721 cells showing good detection .

• Dilution factors: Though the recommended dilution range is 1:200-1:2000 or 1:500-1:1000, validated results have been reported at 1:600 dilution specifically for SH-SY5Y cells .

• Detection system: While specific secondary antibody recommendations aren't detailed in the available data, standard anti-rabbit IgG detection systems are appropriate given the rabbit origin of the primary antibody .

• Expected molecular weight: Researchers should look for a band at approximately 74 kDa, which is the consistently observed molecular weight for ZNF549 .

It's important to note that optimization might be required for different experimental systems, and preliminary titration experiments are recommended when first establishing the Western blot protocol with this antibody.

How should researchers design ZNF549 knockdown experiments?

When designing ZNF549 knockdown experiments, researchers should consider both siRNA and shRNA approaches:

• Available tools: Commercial siRNA/shRNA/RNAi lentivectors specific for human ZNF549 are available and typically come in sets of four different constructs targeting different regions of the ZNF549 transcript .

• Target sequence selection: Since the effectiveness of siRNA can vary depending on the target cell type and experimental conditions, using a set of four different siRNA constructs increases the likelihood of achieving significant knockdown .

• Transduction methods: Researchers can use either viral transduction (using packaged lentivirus) or direct transfection methods (like Lipofectamine) to deliver the siRNA constructs .

• Controls: Appropriate negative controls include scrambled siRNA sequences, which are available for use across human, mouse, and rat experiments .

• Validation: Knockdown efficiency should be assessed using qPCR and/or Western blot with the ZNF549 antibody to confirm protein reduction .

The dual convergent promoter system used in some commercial vectors, where sense and antisense strands of the siRNA are expressed by different promoters rather than in a hairpin loop, helps avoid potential recombination events .

What considerations are important for immunofluorescence studies with ZNF549 antibodies?

For successful immunofluorescence studies of ZNF549, researchers should follow these guidelines:

• Cell selection: A549 cells have been validated for immunofluorescence detection of ZNF549, making them a recommended starting point for new studies .

• Antibody dilution: The recommended dilution range for immunofluorescence is 1:20-1:200, with validated results reported at 1:50 dilution .

• Secondary antibody selection: Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) has been successfully used as a secondary antibody for ZNF549 detection .

• Fixation and permeabilization: While specific fixation protocols aren't detailed in the search results, standard formaldehyde fixation followed by Triton X-100 permeabilization is likely appropriate based on the antibody class.

• Imaging parameters: Researchers should optimize exposure settings to capture the specific subcellular localization of ZNF549 while minimizing background.

When designing multiplexed immunofluorescence experiments, consideration should be given to potential cross-reactivity with other antibodies in the panel.

How should ZNF549 antibodies be stored for optimal performance?

Proper storage of ZNF549 antibodies is critical for maintaining their activity and specificity:

• Storage temperature: ZNF549 antibodies should be stored at -20°C, and aliquoting is generally not recommended for this particular antibody formulation .

• Formulation: The antibody is typically provided in PBS with 0.02-0.1% sodium azide and 50% glycerol at pH 7.3, which helps maintain stability during freeze-thaw cycles .

• Stability: When stored properly, these antibodies remain stable for one year after shipment .

• Working solution preparation: For each experiment, thaw only the amount needed to prepare working dilutions, and keep the stock frozen at -20°C.

• Handling precautions: As with all antibodies containing sodium azide, proper safety precautions should be followed during handling due to the toxic nature of sodium azide.

What controls should be included in ZNF549 antibody experiments?

To ensure reliable and interpretable results when using ZNF549 antibodies, researchers should include the following controls:

• Positive controls: Use cell lines known to express ZNF549, such as SH-SY5Y, A549, HeLa, or SMMC-7721 cells, which have been validated for ZNF549 detection .

• Negative controls: Include samples where the primary antibody is omitted but all other steps are performed identically.

• Isotype controls: Use rabbit IgG at the same concentration as the ZNF549 antibody to identify potential non-specific binding.

• Knockdown validation: For functional studies, comparing antibody detection in wild-type versus ZNF549 knockdown samples can confirm antibody specificity.

• Loading controls: For Western blot applications, include appropriate loading controls such as GAPDH, β-actin, or tubulin to normalize protein loading.

Including these controls in experimental design will strengthen the validity of findings and help troubleshoot any issues that arise.

How can researchers validate ZNF549 antibody specificity in their experimental system?

Validating antibody specificity is critical for accurate data interpretation. For ZNF549 antibodies, consider the following validation approaches:

• Genetic validation: Use ZNF549 knockdown or knockout systems to confirm that the antibody signal decreases or disappears accordingly. Commercial siRNA/shRNA systems targeting ZNF549 can facilitate this approach .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide or recombinant ZNF549 protein before application to samples. Specific signals should be blocked or significantly reduced.

• Multiple antibody validation: If available, compare results using different antibodies against ZNF549 that recognize distinct epitopes.

• Cross-species reactivity analysis: The current ZNF549 antibodies are validated for human samples but not tested in other species . If using in non-human systems, additional validation is essential.

• Molecular weight verification: Always confirm that the detected band appears at the expected molecular weight of 74 kDa in Western blot applications .

What approaches are available for studying ZNF549 function?

Researchers investigating ZNF549 function can employ several complementary approaches:

• Expression studies: Use the validated ZNF549 antibodies for Western blot and immunofluorescence to study expression patterns across different cell types, tissues, or disease states .

• Genetic manipulation: Utilize the available siRNA/shRNA/RNAi lentivectors to knockdown ZNF549 expression and study the resulting phenotypes .

• Protein interaction studies: Consider co-immunoprecipitation with ZNF549 antibodies followed by mass spectrometry to identify interaction partners.

• Chromatin immunoprecipitation (ChIP): As a zinc finger protein, ZNF549 likely binds DNA. ChIP using ZNF549 antibodies could identify genomic binding sites.

• Subcellular localization studies: Immunofluorescence with the validated ZNF549 antibodies can reveal the protein's distribution within cells and potential translocation under various conditions .

These approaches can be combined to build a comprehensive understanding of ZNF549 function in normal and pathological contexts.

What considerations are important when designing ZNF549 overexpression experiments?

When planning ZNF549 overexpression studies, researchers should consider:

• Expression system selection: Choose an expression system compatible with your experimental cell types. Lentiviral vectors similar to those used for knockdown studies may be appropriate for difficult-to-transfect cells .

• Tag selection: Consider adding a tag (e.g., FLAG, HA, GFP) for detection and purification purposes, but position it carefully to avoid interfering with ZNF549 function.

• Expression level control: Use inducible promoters to control expression levels and timing, as overexpression artifacts can occur with constitutive promoters.

• Validation: Confirm successful overexpression using both the tag-specific antibodies and the validated ZNF549 antibodies to ensure the protein is correctly expressed and folded.

• Functional readouts: Design appropriate functional assays based on the hypothesized role of ZNF549 as a transcriptional regulator.

How can researchers integrate ZNF549 studies with broader genomic analyses?

To place ZNF549 research in a broader genomic context, researchers should consider:

• Transcriptomic profiling: Perform RNA-seq after ZNF549 knockdown or overexpression to identify genes regulated by this zinc finger protein.

• Epigenomic integration: Combine ChIP-seq data for ZNF549 binding with histone modification and open chromatin data to understand its role in chromatin regulation.

• Interactome mapping: Use proximity labeling approaches (BioID, APEX) with ZNF549 fusion proteins to identify proteins in its vicinity under different cellular conditions.

• Single-cell analyses: Consider examining ZNF549 expression and function at the single-cell level to identify cell type-specific roles.

• CRISPR screening: Design CRISPR screens to identify genes that interact functionally with ZNF549, potentially revealing synthetic lethal relationships or pathway connections.

These integrative approaches can provide a systems-level understanding of ZNF549 function beyond what can be achieved with antibody-based studies alone.