Recombinant Human ATXN10 Protein: Preparation & Usage Protocol

Recombinant Human ATXN10 Protein is a research-grade recombinant protein that enables scientists to study the molecular functions of ataxin-10, a protein implicated in spinocerebellar ataxia type 10 (SCA10) pathophysiology.

Product Name

Recombinant Human ATXN10 Protein

SKU

rp365072

Protein Origin

Human (Homo sapiens)

Protein Type

Recombinant protein

Primary Application

Biochemical assays, binding studies, functional research on SCA10-related pathways

Recommended Storage

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

Background: ATXN10 and Its Biological Significance

ATXN10 (Ataxin-10) is the protein product of the ATXN10 gene located on human chromosome 22q13.31. It belongs to the HEAT repeat-containing protein superfamily and is ubiquitously expressed in neuronal tissues. ATXN10 plays roles in cell survival, neurite outgrowth, and maintenance of cerebellar Purkinje cells.

Pathogenic intronic AUUCU pentanucleotide repeat expansions within the ATXN10 gene are the causative mutation for spinocerebellar ataxia type 10 (SCA10), an autosomal dominant neurodegenerative disorder predominantly affecting individuals of Latin American ancestry. Research into ATXN10 protein function is therefore critical for understanding both normal cerebellar physiology and the mechanisms underlying SCA10 neurodegeneration.

Recombinant Human ATXN10 Protein (rp365072) provides a well-defined reagent for in vitro studies including protein–protein interaction assays, enzyme-linked immunosorbent assays (ELISA), and structural analyses.

Materials and Equipment Required

• Recombinant Human ATXN10 Protein (rp365072, Aladdin Scientific)
• Sterile, nuclease-free water or recommended reconstitution buffer (e.g., PBS, pH 7.4, with carrier protein such as 0.1% BSA if required)
• Microcentrifuge tubes (low-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Bench-top microcentrifuge
• Dry ice or liquid nitrogen for snap-freezing aliquots
• -80°C ultra-low temperature freezer
• Spectrophotometer or fluorometer for protein quantification (e.g., BCA or Bradford assay reagents)
• SDS-PAGE equipment and Coomassie stain (for purity verification)
• Assay-specific buffers and reagents appropriate to the downstream application

Step-by-Step Reconstitution and Handling Protocol

Step 1: Preparation

Remove the vial from -80°C storage and allow it to equilibrate briefly at room temperature (no longer than 5 minutes) before opening. Do not vortex the vial at any stage.

Step 2: Reconstitution

Reconstitute the lyophilized protein by adding the volume of sterile reconstitution buffer specified in the product datasheet directly to the vial. Gently swirl or pipette up and down until the protein is fully dissolved. If the product is supplied as a liquid, proceed directly to aliquoting.

Step 3: Protein Quantification

Determine the protein concentration using a validated colorimetric method such as BCA or Bradford assay, calibrated against a BSA standard curve. Record the measured concentration for batch traceability.

Step 4: Aliquoting

Divide the reconstituted protein into single-use aliquots in low-binding microcentrifuge tubes to minimize protein loss due to adsorption and to avoid repeated freeze-thaw cycles. Label each aliquot clearly with the date, concentration, and lot number.

Step 5: Storage

Snap-freeze aliquots using dry ice or liquid nitrogen immediately after preparation. Store all aliquots at -80°C. For short-term use within 24–48 hours, aliquots may be kept at 4°C; do not store at 4°C for extended periods as protein stability may be compromised.

Step 6: Purity Verification (Recommended)

Prior to critical experiments, verify protein integrity by running a reducing SDS-PAGE gel. Stain with Coomassie Brilliant Blue and compare the observed band to the expected molecular weight. Confirm purity meets application requirements.

Downstream Application Guidelines

Recombinant Human ATXN10 Protein is suitable for a range of in vitro research applications. The following general guidelines apply:

• Protein–Protein Interaction Studies: Use standard co-immunoprecipitation (co-IP) or pull-down formats. Optimize salt concentration (typically 150 mM NaCl) and detergent conditions to preserve native interactions.
• ELISA / Immunoassays: Use the recombinant protein as a coating antigen or as a positive control. Determine optimal coating concentrations empirically, starting from the range recommended in the product datasheet.
• Western Blot Positive Control: Load an appropriate amount of protein (typically 50–200 ng per lane) alongside experimental samples to confirm antibody specificity and assay performance.
• Structural and Biophysical Studies: Suitable as a starting material for circular dichroism (CD), surface plasmon resonance (SPR), or isothermal titration calorimetry (ITC) once purity and integrity are confirmed.

Always perform a pilot experiment to optimize working concentrations and assay conditions specific to your laboratory setup before conducting large-scale experiments.

Troubleshooting and Quality Control

Issue	Possible Cause	Recommended Action
Protein not dissolving completely	Insufficient buffer volume; protein aggregation	Increase buffer volume; warm gently to room temperature; centrifuge briefly at low speed before use
Lower activity than expected	Repeated freeze-thaw; improper storage temperature	Use freshly thawed single-use aliquots; verify -80°C storage compliance
Unexpected bands on SDS-PAGE	Protein degradation; contamination	Check lot certificate of analysis; use fresh reconstitution buffer; contact Aladdin Scientific technical support
High background in ELISA	Non-specific binding; excess coating concentration	Optimize blocking conditions; titrate coating concentration downward

For persistent issues, contact Aladdin Scientific technical support with your lot number and experimental details.

Frequently asked questions

What is the recommended reconstitution buffer for Recombinant Human ATXN10 Protein?

The reconstitution buffer and volume are specified in the product-specific datasheet supplied with each lot. As a general guideline, sterile PBS (pH 7.4) supplemented with 0.1% BSA is commonly used for recombinant proteins to maintain stability; however, always refer to the official datasheet for lot-specific instructions.

How should Recombinant Human ATXN10 Protein be stored to maintain stability?

Store the protein at -80°C in single-use aliquots. Avoid repeated freeze-thaw cycles, as these can lead to protein aggregation and loss of activity. For experiments scheduled within 24–48 hours, a single aliquot may be kept at 4°C, but longer storage at this temperature is not recommended.

Which research applications is Recombinant Human ATXN10 Protein suitable for?

Recombinant Human ATXN10 Protein (rp365072) is suitable for in vitro applications including protein–protein interaction studies (co-IP, pull-down), ELISA as a coating antigen or positive control, Western blot positive control, and biophysical studies such as SPR or ITC. Suitability for specific applications should be confirmed by pilot experiments.

How can I verify the purity and integrity of the protein before use?

Run a reducing SDS-PAGE gel with Coomassie Brilliant Blue staining and compare the band position with the expected molecular weight stated in the datasheet. A single dominant band at the correct molecular weight indicates acceptable purity and integrity. The certificate of analysis (CoA) provided with the product also contains lot-specific purity data.

What is the biological relevance of ATXN10 in disease research?

ATXN10 encodes ataxin-10, a HEAT repeat protein involved in neuronal survival and cerebellar function. Pathogenic expansions of an intronic AUUCU pentanucleotide repeat in the ATXN10 gene cause spinocerebellar ataxia type 10 (SCA10), an autosomal dominant neurodegenerative disease. Research using recombinant ATXN10 protein helps elucidate its normal cellular functions and the mechanisms by which dysfunction leads to neurodegeneration.

References

1. Teive HAG, Roa BB, Raskin S, et al. Clinical phenotype of Brazilian families with spinocerebellar ataxia 10. Neurology. 2004;63(8):1509-1512.
2. Matsuura T, Yamagata T, Burgess DL, et al. Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10. Nature Genetics. 2000;26(2):191-194.
3. Aladdin Scientific. Recombinant Human ATXN10 Protein (rp365072) Product Datasheet. Aladdin Scientific.

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