RNF14 Mouse mAb: Selection, Application & Protocol Guide

RNF14 Mouse mAb is a mouse-derived monoclonal antibody specifically targeting the RNF14 E3 ubiquitin ligase protein, supplied in PBS formulation and validated for use in key immunological detection applications.

Product SKU

Ab351055

Antibody Type

Mouse Monoclonal Antibody (mAb)

Target Protein

RNF14 (Ring Finger Protein 14), an E3 ubiquitin ligase

Formulation

PBS (Phosphate-Buffered Saline), no additional additives stated

Host Species

Mouse

Reference Source

absea #KC-46127

Overview of RNF14 and Its Biological Significance

RNF14 (Ring Finger Protein 14), also known as ARA54, is a member of the RING finger domain-containing E3 ubiquitin ligase family. E3 ubiquitin ligases play a central role in the ubiquitin-proteasome system (UPS) by facilitating the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins, thereby tagging them for proteasomal degradation or altering their activity and interactions.

RNF14 has been reported to interact with androgen receptor (AR) signaling pathways and is implicated in transcriptional regulation. Research interest in RNF14 spans oncology, cell cycle regulation, and protein quality control. A validated, highly specific monoclonal antibody such as Ab351055 is therefore an essential reagent for investigating RNF14 expression levels, protein–protein interactions, and subcellular localization in these contexts.

Antibody Specifications and Formulation Guidance

Ab351055 is a mouse monoclonal antibody raised against RNF14. The antibody is supplied in PBS (phosphate-buffered saline) without additional carrier proteins or preservatives stated in the source material. Researchers should note the following handling recommendations for PBS-only formulated antibodies:

• Storage: Store at –20 °C for long-term storage. Avoid repeated freeze–thaw cycles. Aliquot upon first use to preserve antibody activity.
• Carrier protein: Because no BSA or glycerol stabilizer is indicated, consider adding 50% glycerol (final concentration) before long-term freezing to enhance stability, unless downstream applications are sensitive to glycerol.
• Concentration: Refer to the Certificate of Analysis (CoA) supplied with each lot for confirmed protein concentration and titer information.
• Sterility: Handle under aseptic conditions if the antibody is to be used in cell-based assays.

Always consult the product datasheet (absea #KC-46127) and the lot-specific CoA before use.

Recommended Application Protocols

While specific validated applications should be confirmed on the product datasheet, the following general protocols represent best practices for mouse monoclonal antibodies targeting intracellular E3 ubiquitin ligases such as RNF14:

Western Blot (WB)

1. Lyse cells or tissue in RIPA buffer supplemented with protease inhibitors. Centrifuge at 12,000 × g for 15 min at 4 °C.
2. Determine protein concentration (BCA or Bradford assay). Load 20–40 µg total protein per lane on SDS-PAGE.
3. Transfer to PVDF membrane. Block with 5% non-fat milk in TBST for 1 hour at room temperature.
4. Incubate with RNF14 Mouse mAb (Ab351055) at the manufacturer-recommended dilution (see CoA) overnight at 4 °C.
5. Wash 3× with TBST (10 min each). Incubate with HRP-conjugated anti-mouse secondary antibody for 1 hour at RT.
6. Detect by ECL chemiluminescence. Expected band size for RNF14: approximately 50–60 kDa (verify with CoA).

Immunoprecipitation (IP)

1. Pre-clear lysate with Protein G or Protein A beads. Add Ab351055 at recommended concentration and incubate overnight at 4 °C with rotation.
2. Add fresh beads and incubate for 2 hours. Wash beads 4× with lysis buffer. Elute with SDS sample buffer and analyze by WB.

Immunofluorescence (IF)

1. Fix cells with 4% paraformaldehyde for 15 min. Permeabilize with 0.1% Triton X-100 for 10 min.
2. Block with 5% normal goat serum for 1 hour. Incubate with primary antibody at recommended dilution overnight at 4 °C.
3. Wash and incubate with fluorophore-conjugated anti-mouse secondary antibody. Counterstain nuclei with DAPI. Image by confocal or fluorescence microscopy.

Troubleshooting Common Issues

The following troubleshooting table addresses common issues encountered when working with RNF14 Mouse mAb (Ab351055):

Issue	Possible Cause	Recommended Action
No signal on WB	Antibody degraded; incorrect dilution; antigen masked	Check storage conditions; titrate antibody; optimize lysis conditions. Confirm RNF14 expression in chosen cell line.
High background	Insufficient blocking; secondary antibody cross-reactivity; high primary antibody concentration	Extend blocking time; increase TBST wash steps; reduce primary antibody concentration.
Multiple non-specific bands	Protein degradation; over-exposure	Add protease inhibitors to lysis buffer; reduce ECL exposure time.
Weak IF signal	Over-fixation; inadequate permeabilization	Optimize fixation time; increase Triton X-100 concentration or duration.

Frequently asked questions

What is the recommended storage condition for RNF14 Mouse mAb (Ab351055)?

Ab351055 is formulated in PBS and should be stored at –20 °C for long-term preservation. To maintain antibody integrity, avoid repeated freeze–thaw cycles by preparing single-use aliquots upon first thaw. Consult the lot-specific Certificate of Analysis for any additional storage recommendations.

Which experimental applications is RNF14 Mouse mAb validated for?

The validated applications for Ab351055 should be confirmed on the official product datasheet (absea #KC-46127). Typical applications for anti-RNF14 monoclonal antibodies include Western blot (WB), immunoprecipitation (IP), and immunofluorescence (IF). Always verify suitability for your specific application with the CoA before proceeding.

What is the expected molecular weight of RNF14 detected by this antibody?

RNF14 is expected to appear at approximately 50–60 kDa on Western blot; however, post-translational modifications such as ubiquitination or phosphorylation may cause band shifts. The exact expected molecular weight should be confirmed with the product datasheet and CoA for Ab351055.

Can RNF14 Mouse mAb be used for flow cytometry or ELISA?

The suitability of Ab351055 for flow cytometry or ELISA has not been confirmed in the available source material. Researchers interested in these applications should contact Aladdin Scientific technical support for guidance or consult the product datasheet for a complete list of validated applications.

Is RNF14 Mouse mAb cross-reactive with non-human species?

Species cross-reactivity information for Ab351055 is not explicitly stated in the available source material. Please refer to the official product datasheet (absea #KC-46127) or contact Aladdin Scientific for confirmed cross-reactivity data before using this antibody in non-human model systems.

References

1. absea product datasheet KC-46127: RNF14 Mouse Monoclonal Antibody. absea Biotechnology Ltd.
2. Hershko A, Ciechanover A. The ubiquitin system. Annu Rev Biochem. 1998;67:425–479. doi:10.1146/annurev.biochem.67.1.425
3. Ito H, Fukuda Y, Murata K, Kimura A. Ring finger protein 14 (RNF14/ARA54) as a coactivator of androgen receptor signaling. Published literature — specific citation details not available in source material.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.