SRRM4 Antibody, FITC conjugated

What is the biological significance of SRRM4 in neural cell differentiation?

SRRM4, or Serine/arginine repetitive matrix protein 4, is a splicing factor specifically required for neural cell differentiation. It acts in conjunction with nPTB/PTBP2 by binding directly to regulated target transcripts, promoting neural-specific exon inclusion in genes critical for neural development. For instance, SRRM4 facilitates the inclusion of exon 10 in nPTB/PTBP2 transcripts, thereby increasing the expression of neural-specific nPTB/PTBP2. Additionally, it promotes exon 16 inclusion in DAAM1 within neuron extracts . These mechanisms underscore its pivotal role in the regulation of alternative splicing events essential for neuronal function.

How can SRRM4 antibody conjugated with FITC be utilized in experimental design?

The FITC-conjugated SRRM4 antibody is particularly suited for fluorescence-based applications such as flow cytometry and immunofluorescence microscopy. Researchers can use this antibody to visualize SRRM4 expression patterns in neuronal tissues or cultured cells under a fluorescence microscope. The excitation wavelength of FITC at 488 nm and emission at 520 nm ensures compatibility with standard blue laser systems used in flow cytometers . Experimental designs should include proper controls such as isotype controls and non-conjugated antibodies to validate specificity.

What are the methodological considerations for using SRRM4 antibody in immunoprecipitation assays?

Immunoprecipitation assays using SRRM4 antibodies are instrumental in studying protein-protein interactions and RNA binding events mediated by SRRM4. For instance, researchers have employed FLAG-tagged SRRM4 constructs to perform RNA immunoprecipitation (RIP) assays to identify its binding to specific motifs like the UGC sequence within protrudin pre-mRNA . Key considerations include optimizing antibody concentrations, ensuring the quality of nuclear extracts, and validating results through complementary techniques such as RT-PCR and Western blot analysis.

How does SRRM4 regulate alternative splicing mechanisms?

SRRM4 regulates alternative splicing by recognizing specific motifs within pre-mRNA sequences. For example, it binds to atypical acceptor sequences flanking exon L of protrudin pre-mRNA, promoting exon L inclusion in neuronal cells while repressing it in non-neuronal cells due to the absence of SRRM4 . This regulation involves interactions with splicing factors like U2AF65 and U2AF35 at polypyrimidine tracts and splice sites. Knockdown experiments have shown that depletion of SRRM4 reduces exon inclusion for known targets such as Mef2a and Synj1 transcripts .

What challenges might arise when interpreting data from SRRM4 knockdown or overexpression studies?

Knockdown or overexpression studies can yield complex data due to compensatory mechanisms within cellular systems. For instance, depletion of SRRM4 reduces the abundance of protrudin-L mRNA while increasing protrudin-S mRNA levels in Neuro2A cells . Overexpression studies may enhance exon inclusion across various targets but could also lead to off-target effects or saturation of splicing machinery. Researchers should employ rigorous controls and replicate experiments across different cell lines to ensure reproducibility.

How can flow cytometry be optimized for detecting SRRM4 using FITC-conjugated antibodies?

Flow cytometry detection of SRRM4 requires careful optimization of antibody titration and staining protocols. The recommended concentration is typically less than or equal to 0.25 µg per test, with cell numbers ranging from $10^5$ to $10^8$ per sample . Researchers should empirically determine optimal staining conditions based on cell type and experimental requirements while ensuring proper gating strategies to exclude debris or non-specific signals.

What are the implications of post-translational modifications on SRRM4 function?

Post-translational modifications such as phosphorylation can significantly impact SRRM4's activity and interactions with other molecules. These modifications may alter its ability to bind target transcripts or recruit additional splicing factors . Advanced techniques like mass spectrometry can be employed to identify specific modification sites and assess their functional consequences.

How does the subcellular localization of SRRM4 influence its role in splicing?

SRRM4 is predominantly localized within the nucleus where splicing occurs . Its nuclear presence ensures access to pre-mRNA substrates and interaction with other components of the spliceosome complex. Immunocytochemistry using FITC-conjugated antibodies can be utilized to confirm nuclear localization patterns under different experimental conditions.

What experimental controls are necessary when using FITC-conjugated SRRM4 antibodies?

Effective controls include using an isotype-matched antibody conjugated with FITC to account for non-specific binding, as well as employing secondary antibodies conjugated with different fluorophores for multiplexing experiments . Additionally, researchers should validate antibody specificity through Western blotting or immunohistochemistry using known positive and negative samples.

Can SRRM4-dependent splicing events be quantitatively analyzed? If so, how?

Quantitative analysis of SRRM4-dependent splicing events can be achieved through RT-qPCR targeting specific exons regulated by SRRM4 (e.g., exon L of protrudin transcripts). High-throughput RNA sequencing offers a more comprehensive approach by identifying global changes in alternative splicing patterns upon manipulation of SRRM4 levels .