Recombinant Human ELOA Protein: Handling & Usage Protocol

Recombinant Human ELOA Protein is a research-grade recombinant protein representing the human transcription elongation factor A (ELOA), supplied by Aladdin Scientific to support biochemical characterization, protein–protein interaction studies, and transcription-related functional assays.

Product Name

Recombinant Human ELOA Protein

SKU

rp365273

Protein Function

Transcription elongation factor A; involved in regulation of RNA polymerase II-mediated transcription elongation

Form

Recombinant protein (lyophilized or liquid; confirm with datasheet)

Recommended Storage

Store at -80°C; avoid repeated freeze-thaw cycles

Application

Biochemical assays, protein interaction studies, transcription elongation research

Background: ELOA and Transcription Elongation

Elongin A (ELOA, also known as Transcription Elongation Factor B Polypeptide 3 or TCEB3) is a subunit of the Elongin (SIII) complex, which functions as a positive regulator of RNA polymerase II (RNAPII) transcription elongation. The Elongin complex, comprising Elongin A, Elongin B, and Elongin C subunits, stimulates the overall rate of elongation by suppressing transient pausing of RNAPII at many sites along the DNA template. ELOA is the transcriptionally active subunit of the complex and is essential for productive mRNA synthesis. Additionally, Elongin A participates in ubiquitin ligase complexes that target stalled RNAPII for proteasomal degradation, linking transcription quality control to protein degradation pathways. Research into ELOA is relevant to understanding gene regulation, DNA damage response, and transcription-coupled repair mechanisms.

Materials Required

• Recombinant Human ELOA Protein (SKU: rp365273, Aladdin Scientific)
• Sterile, nuclease-free water or reconstitution buffer as specified on the product datasheet
• Phosphate-buffered saline (PBS, pH 7.4) or assay-compatible buffer
• Carrier protein (e.g., 0.1% BSA) for dilutions if required
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Pipettes and calibrated tips
• Ice bucket and dry ice for cold-chain handling
• SDS-PAGE or Western blot reagents (for QC verification)
• Relevant assay reagents (per downstream application)

Step-by-Step Protocol

1. Receipt and Initial Inspection

Upon receipt, immediately transfer the Recombinant Human ELOA Protein to the recommended storage temperature (-80°C for long-term storage). Inspect the vial for integrity and verify lot number and expiry date against the Certificate of Analysis (CoA).

2. Reconstitution

Centrifuge the vial briefly (1,000 × g, 30 seconds) before opening to collect lyophilized material at the bottom. Reconstitute the protein by adding sterile, nuclease-free water or the buffer specified in the product datasheet to achieve the recommended concentration (refer to CoA for exact volume). Gently pipette to mix; do not vortex. Allow to sit on ice for 5–10 minutes to ensure complete dissolution.

3. Aliquoting

Once reconstituted, prepare single-use aliquots in low-binding microcentrifuge tubes to avoid repeated freeze-thaw cycles, which can lead to protein aggregation and loss of activity. Label each aliquot with protein identity, concentration, date, and lot number.

4. Storage

Store short-term working aliquots at 4°C for up to 1 week. For long-term storage, snap-freeze aliquots in liquid nitrogen or on dry ice and store at -80°C. Addition of a carrier protein (0.1% BSA) to diluted working stocks is recommended to maintain stability at low concentrations.

5. Quality Control Verification

Prior to experimental use, verify protein integrity by SDS-PAGE under reducing conditions. Confirm purity and molecular weight match values stated on the CoA. Western blot with an anti-ELOA antibody may be performed to confirm protein identity.

6. Functional Application

Use the reconstituted protein at concentrations determined by preliminary titration experiments. Common applications include in vitro transcription elongation assays, pull-down or co-immunoprecipitation (co-IP) assays to study Elongin complex formation, and ubiquitin ligase activity assays. Follow validated assay protocols and include appropriate positive and negative controls.

Troubleshooting

Problem	Possible Cause	Recommended Action
Protein precipitation after reconstitution	Incorrect buffer, rapid temperature change, or vortexing	Use recommended buffer at recommended temperature; mix gently by pipetting; centrifuge briefly and use supernatant
Low or absent activity in functional assay	Protein denaturation due to improper storage or freeze-thaw cycling	Use fresh aliquot; verify storage conditions; add BSA carrier; check CoA for activity specifications
Unexpected band size on SDS-PAGE	Protein degradation or aggregation; unexpected post-translational modifications	Compare with CoA molecular weight; use fresh aliquot; check reducing vs. non-reducing conditions
High background in binding assays	Non-specific interactions; inadequate blocking	Optimize blocking conditions; titrate protein concentration; include appropriate controls

Safety and Disposal

Recombinant Human ELOA Protein is intended for research use only and is not approved for diagnostic or therapeutic applications. Handle all recombinant proteins following your institution's biosafety guidelines. Wear appropriate personal protective equipment (PPE) including gloves and a lab coat. Dispose of protein solutions and contaminated materials according to local regulations for biological waste. Refer to the Safety Data Sheet (SDS) provided with the product for specific hazard information.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human ELOA Protein?

The reconstitution buffer is specified on the product-specific Certificate of Analysis (CoA) and datasheet included with each lot. Generally, sterile PBS (pH 7.4) supplemented with 0.1% BSA is suitable for many recombinant proteins; however, always refer to the CoA for lot-specific recommendations to ensure optimal protein activity and stability.

How should I store Recombinant Human ELOA Protein to maintain its activity?

For long-term storage, keep the protein at -80°C in single-use aliquots to avoid repeated freeze-thaw cycles. Short-term working stocks may be stored at 4°C for up to one week. Adding a carrier protein such as 0.1% BSA to diluted stocks can help maintain stability at low concentrations. Avoid exposing the protein to repeated temperature fluctuations.

What applications is Recombinant Human ELOA Protein suitable for?

Recombinant Human ELOA Protein is suitable for biochemical applications including in vitro transcription elongation assays, protein–protein interaction studies (e.g., pull-down assays, co-immunoprecipitation) to investigate Elongin complex assembly, and ubiquitin ligase activity assays. It may also be used as an antigen for antibody validation. Always confirm suitability for your specific application against the product datasheet.

How can I verify the quality and identity of the protein upon receipt?

Upon receipt, perform SDS-PAGE under reducing conditions and compare the observed band to the molecular weight stated on the CoA. For identity confirmation, Western blot analysis using a validated anti-ELOA antibody is recommended. Ensure purity and band pattern match CoA specifications before proceeding with functional experiments.

References

1. Bradsher J, et al. Molecular characterization of the ELL (eleven-nineteen lysine-rich leukemia) family of RNA polymerase II elongation factors. J Biol Chem. 2000.
2. Aso T, et al. Elongin (SIII): a multisubunit regulator of elongation by RNA polymerase II. Science. 1995;269(5229):1439-1443.
3. Yasukawa T, et al. Mammalian Elongin A complex mediates DNA-damage-induced ubiquitylation and degradation of Rpb1. EMBO J. 2008;27(24):3256-3266.

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