SRS5 Antibody

FAQs for SRS5 Antibody in Academic Research

SRS5 (Serine/arginine-rich splicing factor 5) is a critical regulator of alternative pre-mRNA splicing. Below are methodologically focused FAQs addressing both foundational and advanced research challenges, synthesized from peer-reviewed studies and antibody validation data.

How to validate SRS5 antibody specificity in Western blot (WB)?

• Method: Use knockout (KO) cell lines (e.g., HEK-293T SRSF5-KO [ab266246]) as negative controls. Compare lysates from wild-type and KO cells under reducing conditions.

  • Example: ab67175 shows a 40 kDa band in wild-type HEK-293T lysates (vs. predicted 31 kDa), with loss of signal in KO lysates .

  • Troubleshooting: Optimize blocking buffers (e.g., fluorescent WB blocking solution) and secondary antibodies (e.g., IRDye® 800CW/680RD) .

What are the recommended applications and dilutions for SRS5 antibodies?

• Applications:

  Application	Antibody (Clone)	Dilution	Sample Type
  WB	ab67175 (Mouse)	1:500	Human, Mouse
  WB	16237-1-AP (Rabbit)	1:500–1:1000	Human, Mouse, Rat
  ICC/IF	ab67175	1 µg/mL	HeLa cells

• Notes: Titrate antibodies empirically. For ICC/IF, use 4% formaldehyde fixation and permeabilization with 0.3M glycine/1% BSA .

Why is there a discrepancy between observed and predicted molecular weights?

• Observed vs. Predicted:

  Antibody	Predicted (kDa)	Observed (kDa)	Possible Reasons
  ab67175	31	40	Post-translational modifications (e.g., phosphorylation)
  16237-1-AP	31	34–40	Alternative splicing isoforms

How to design experiments investigating SRS5’s role in alternative splicing?

• Approach:

  • Knockdown/Overexpression: Use siRNA or CRISPR-Cas9 in cell models (e.g., HeLa, A549) .

  • RNA-Seq: Pair with RIP-Seq (RNA Immunoprecipitation) to identify SRS5-bound pre-mRNA targets .

  • Functional Validation: Assess splicing outcomes via RT-PCR or minigene reporters .

How to resolve contradictions in antibody performance across studies?

• Case Example: A study reports weak SRS5 signal in HepG2 lysates , while another detects strong expression .

  • Solutions:

    • Buffer Optimization: Adjust lysis buffer composition (e.g., inclusion of phosphatase inhibitors).

    • Cross-Validation: Use orthogonal methods (e.g., IHC in formalin-fixed tissues vs. ELISA ).

    • Epitope Mapping: Compare immunogen sequences (e.g., ab67175 vs. 16237-1-AP target different epitopes) .

What functional assays confirm SRS5’s role in metabolic or disease pathways?

• Published Findings:

  • Metabolism: SRS5 regulates iron-sulfur cluster biogenesis in hematopoietic stem cells via splicing of Aspartyl-tRNA synthetase 2 transcripts .

  • Disease Models:

    • Cancer: NEAT1 lncRNA recruits SRS5 to promote breast cancer metastasis by altering splicing of metabolic genes .

    • Fibrosis: SRS5 drives alternative splicing of TGF-β1 in pleural fibrosis .

• Methods: Use siRNA-mediated knockdown followed by RNA-Seq and metabolomics (e.g., LC-MS) .

Methodological Best Practices

• Antibody Selection:

  Criterion	Polyclonal (16237-1-AP)	Monoclonal (ab67175)
  Advantages	Broad epitope recognition	Batch-to-batch consistency
  Limitations	Risk of cross-reactivity	Limited to specific isoforms

• Cross-Reactivity Testing:

  • Perform immunogen sequence alignment (e.g., UniProt Q13243).

  • Pre-adsorb antibodies with blocking peptides in competitive ELISA .