SRP9 Antibody

Introduction to SRP9 Antibody

SRP9 antibodies are immunoglobulins specifically designed to recognize and bind to the Signal Recognition Particle 9kDa protein (SRP9). These antibodies serve as essential research tools for investigating the expression, localization, and function of SRP9 across various experimental settings. Commercial SRP9 antibodies are available in both polyclonal and monoclonal formats, derived primarily from rabbit and mouse hosts, and can be obtained in various conjugated and unconjugated forms to accommodate different research applications .

The target protein, SRP9, is a component of the signal recognition particle (SRP) complex, a ribonucleoprotein assembly that mediates cotranslational targeting of secretory and membrane proteins to the endoplasmic reticulum. SRP9 specifically forms a heterodimer with SRP14, constituting the elongation arrest domain of SRP, which is essential for proper protein synthesis and cellular function .

Post-Translational Modifications of SRP9

SRP9 undergoes various post-translational modifications that influence its activity and interactions. These modifications have been documented and are summarized in the following table:

These modifications highlight the complex regulation of SRP9 function and may serve as targets for future therapeutic interventions .

Biological Role and Significance

Signal-recognition-particle assembly plays a crucial role in targeting secretory proteins to the rough endoplasmic reticulum membrane . SRP9, together with SRP14 and the Alu portion of the SRP RNA, constitutes the elongation arrest domain of SRP. This domain is responsible for pausing translation until the ribosome-nascent chain complex reaches the endoplasmic reticulum, ensuring proper protein localization and folding .

Antibody Formats and Conjugations

SRP9 antibodies are available in various formats to accommodate different research applications:

1. Unconjugated primary antibodies for general use

2. HRP-conjugated for direct detection in Western blotting

3. Fluorescent conjugates (FITC, PE, various Alexa Fluor dyes) for direct visualization in immunofluorescence

4. Agarose-conjugated for immunoprecipitation applications

These diverse formats enhance flexibility in experimental design and detection methods .

Western Blotting

Western blotting represents one of the most common applications for SRP9 antibodies, allowing detection of the approximately 10 kDa SRP9 protein in cell and tissue lysates. Typical recommended dilutions range from 1:500 to 1:2000, although optimal concentrations should be determined experimentally for each antibody and sample type .

Published studies have successfully detected SRP9 in various human cell lines including HeLa, HepG2, A2780, MCF-7, SKOV-3, and HuH-7, as well as in mouse tissue samples .

Immunohistochemistry and Immunocytochemistry

SRP9 antibodies have been effectively employed for immunohistochemical (IHC) and immunocytochemical (ICC) detection of SRP9 in fixed tissue sections and cultured cells, respectively. For IHC applications, recommended dilutions typically range from 1:50 to 1:200, while ICC applications often utilize dilutions between 1:100 and 1:500 .

Successful IHC detection has been reported in human prostate cancer tissue, with antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) enhancing detection quality .

Immunoprecipitation and ELISA

Selected SRP9 antibodies support immunoprecipitation (IP) applications, allowing isolation of SRP9 and its interacting partners from complex protein mixtures. Recommended antibody amounts for IP typically range from 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate .

ELISA applications have also been documented, enabling quantitative measurement of SRP9 levels in biological samples .

Recommended Dilutions for Various Applications

The following table provides general guidance for SRP9 antibody dilutions across common applications:

It is important to note that optimal dilutions may vary between antibody preparations and should be determined experimentally for each specific application and sample type .

SRP9 in Cancer Research

Immunohistochemical analysis using SRP9 antibodies has revealed that SRP9 can be preferentially expressed in the nucleus of cancerous regions in some cases, while this nuclear localization is minimal in others. This differential localization pattern suggests potential functional implications beyond SRP9's canonical role in protein targeting .

SRP9 in Neurological Disorders

A groundbreaking study identified SRP9 as a febrile seizure susceptibility gene through a phenotype-driven genetic strategy in mice . The research revealed that mice with reduced SRP9 expression exhibited lower febrile seizure susceptibility, suggesting a potential mechanistic link between SRP9 levels and neuronal excitability .

Further investigation demonstrated that mice with reduced SRP9 expression displayed reduced hippocampal AMPA and NMDA currents, and downregulation of neuronal SRP9 reduced surface expression of AMPA receptor subunit GluA1. These findings indicate that SRP9 may convey its effects through endoplasmic reticulum-dependent synthesis and trafficking of membrane proteins, including glutamate receptors .

Clinical relevance was established by the observation that mesial temporal lobe epilepsy (mTLE) patients with antecedent febrile seizures had higher SRP9 expression than patients without such history. Additionally, an SRP9 promoter SNP (rs12403575(G/A)) was genetically associated with febrile seizures and mTLE, highlighting SRP9's potential role in epileptogenesis .

SRP9 Localization to Stress Granules

Intriguing research has revealed that SRP9, unlike other SRP components, can localize to stress granules following arsenite treatment in both human and mouse cells . Double immunofluorescence staining experiments showed that while SRP9/14 localized to granular structures together with FMRP (a stress granule marker) after arsenite treatment, other SRP components (SRP19, SRP68, SRP72) failed to colocalize with these structures .

This selective localization suggests that SRP9/14, when present in stress granules, is not part of the complete SRP complex but may serve alternative functions in stress response. Furthermore, knockdown of SRP9/14 affected stress granule size, indicating a potential regulatory role in stress granule assembly or dynamics .

Antibody Production and Purification

SRP9 antibodies are typically generated by immunizing host animals (primarily rabbits for polyclonal antibodies and mice for monoclonal antibodies) with synthetic peptides derived from human SRP9 protein. Common immunogens include peptides corresponding to internal regions or spanning from the N-terminus to various points within the sequence .

Purification methods vary by manufacturer but frequently employ affinity chromatography techniques. For example, one supplier notes that "the antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin" , while another states their antibody underwent "antigen affinity purification" .

Validation and Quality Control

Manufacturers employ various methods to validate SRP9 antibodies, including:

1. Western blot analysis using cell lines known to express SRP9 (e.g., HeLa, HepG2)

2. Testing across multiple applications (WB, IHC, IF, IP) to confirm versatility

3. Cross-reactivity testing with samples from multiple species

4. Specificity confirmation through peptide competition or knockdown experiments

These validation steps ensure the antibodies specifically recognize SRP9 and perform consistently across applications .