Recombinant Human FARSB Protein: Handling & Application Protocol

Recombinant Human FARSB Protein is the recombinantly expressed beta subunit of human phenylalanyl-tRNA synthetase, designed to support biochemical studies of aminoacylation, protein–protein interactions, and related cellular processes.

Product Name

Recombinant Human FARSB Protein

SKU

rp367640

Protein Function

Beta subunit of phenylalanyl-tRNA synthetase; involved in aminoacylation of tRNA with phenylalanine

Recommended Storage

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

Typical Applications

Aminoacylation assays, protein interaction studies, SDS-PAGE/Western blot verification

Host / Expression System

Refer to product datasheet for confirmed expression system details

Materials and Equipment Required

Before beginning, ensure the following materials and equipment are available:

• Recombinant Human FARSB Protein (SKU: rp367640), thawed on ice
• Sterile, nuclease-free water or reconstitution buffer as specified on the product datasheet
• Microcentrifuge tubes (1.5 mL, low-protein-binding recommended)
• Calibrated micropipettes and sterile tips
• Refrigerated microcentrifuge
• SDS-PAGE or Western blot apparatus for quality control
• Relevant assay buffers (e.g., aminoacylation reaction buffer: 100 mM HEPES pH 7.5, 10 mM MgCl₂, 2 mM ATP, 0.1 mM phenylalanine)
• Protein quantification reagents (e.g., BCA or Bradford assay kit)

Note: Always consult the product-specific datasheet supplied with SKU rp367640 for confirmed buffer compositions and concentration values.

Reconstitution and Preparation

Follow these steps to reconstitute and prepare the Recombinant Human FARSB Protein for downstream applications:

1. Thaw: Remove the vial from -80 °C storage and thaw slowly on ice for 15–30 minutes. Do not thaw at room temperature or use a water bath.
2. Centrifuge: Briefly spin the vial (1,000 × g, 30 seconds) to collect any condensate from the cap and walls.
3. Reconstitute: Add the volume of sterile reconstitution buffer specified in the product datasheet directly to the lyophilized pellet (if supplied lyophilized). Gently pipette up and down; do not vortex, as this may denature the protein.
4. Equilibrate: Allow the reconstituted solution to stand on ice for 5 minutes to ensure complete dissolution.
5. Quantify: Measure protein concentration using a BCA or Bradford assay. Compare to the nominal concentration stated on the Certificate of Analysis.
6. Aliquot: Divide into single-use aliquots to prevent repeated freeze-thaw cycles. Label each aliquot with date and concentration.
7. Store: Snap-freeze aliquots in liquid nitrogen and store at -80 °C. For short-term use within 1 week, store at 4 °C with a carrier protein (e.g., 0.1% BSA) if recommended by the datasheet.

Critical: Repeated freeze-thaw cycles significantly reduce protein activity. Prepare aliquots sized for single experimental use.

Quality Control Verification

Verify the integrity and purity of Recombinant Human FARSB Protein before use in functional assays:

• SDS-PAGE: Run 1–2 µg of the reconstituted protein on a 10–12% SDS-PAGE gel under reducing conditions. A single dominant band at the expected molecular weight confirms purity. Consult the datasheet for the confirmed molecular weight of the recombinant construct.
• Western Blot: Transfer to PVDF or nitrocellulose membrane and probe with a validated anti-FARSB antibody or an antibody against the recombinant tag (e.g., His-tag). Confirm band position corresponds to expected molecular weight.
• Protein Quantification: Verify that measured concentration is within ±15% of the nominal value stated in the Certificate of Analysis.
• Endotoxin Check: If the protein is to be used in cell-based or in vivo assays, confirm endotoxin levels are below the threshold specified in the datasheet (typically <1.0 EU/µg).

Flag: Exact molecular weight and purity percentage should be confirmed from the product Certificate of Analysis for SKU rp367640. Values not independently verified here.

Functional Application: Aminoacylation Assay

FARSB, as the beta subunit of phenylalanyl-tRNA synthetase (PheRS), plays a structural and catalytic auxiliary role in the aminoacylation of tRNA^Phe. The following outlines a general aminoacylation activity assay framework:

1. Prepare reaction mixture: Combine FARSB protein with its catalytic partner subunit (FARSA, the alpha subunit) at the molar ratio specified in the relevant literature or datasheet. Prepare in aminoacylation buffer (e.g., 100 mM HEPES pH 7.5, 10 mM MgCl₂, 2 mM ATP, 0.1 mM phenylalanine, 0.1 mg/mL BSA).
2. Add substrate tRNA: Include purified tRNA^Phe substrate at the concentration recommended by validated references.
3. Incubate: Incubate the reaction at 37 °C for the time course appropriate to your experimental design (e.g., 10–60 minutes).
4. Terminate reaction: Stop by addition of cold TCA (trichloroacetic acid) or by transfer to ice, as appropriate for your detection method.
5. Detect: Measure aminoacylation using radiolabeled [¹⁴C]-phenylalanine incorporation into tRNA (filter-binding assay) or an equivalent non-radioactive method.

For protein–protein interaction studies, FARSB can be used in pull-down assays, surface plasmon resonance (SPR), or co-immunoprecipitation experiments using standard protocols.

Note: Specific optimal concentrations, incubation times, and buffer compositions should be validated empirically and cross-referenced with current literature.

Troubleshooting

Problem	Possible Cause	Recommended Action
No or low protein activity	Protein denatured during thaw or handling	Ensure thawing on ice; avoid vortexing; check freeze-thaw cycle count
Multiple bands on SDS-PAGE	Protein degradation or aggregation	Confirm storage at -80 °C; add protease inhibitors; use fresh aliquot
Low yield after reconstitution	Incomplete dissolution or protein loss to tube walls	Use low-binding tubes; allow adequate dissolution time; add carrier protein (BSA)
High background in Western blot	Non-specific antibody binding or excess protein loaded	Optimize antibody dilution; reduce protein load; increase blocking time
Inconsistent aminoacylation signal	Suboptimal FARSA:FARSB ratio or buffer composition	Titrate subunit ratio; verify buffer pH and ATP freshness; include positive control

Frequently asked questions

What is the biological function of FARSB?

FARSB encodes the beta subunit of phenylalanyl-tRNA synthetase (PheRS), a heterotetrameric aminoacyl-tRNA synthetase responsible for charging tRNA with phenylalanine during protein synthesis. The beta subunit contributes to the structural integrity of the holoenzyme complex and supports catalytic activity of the alpha subunit (FARSA).

How should I store the Recombinant Human FARSB Protein to maintain activity?

Store the protein at -80 °C in single-use aliquots to prevent activity loss from repeated freeze-thaw cycles. For short-term use (up to one week), storage at 4 °C may be acceptable if a stabilizing carrier protein such as 0.1% BSA is included, as specified in the product datasheet. Always consult the Certificate of Analysis for lot-specific storage recommendations.

Can FARSB protein be used alone in aminoacylation assays?

No. FARSB is the non-catalytic beta subunit and requires co-assembly with the catalytic alpha subunit FARSA to form the active PheRS heterotetrameric complex. For aminoacylation activity assays, both subunits must be present at an appropriate molar ratio. Researchers should source FARSA separately and pre-assemble the complex under conditions validated in peer-reviewed literature.

What quality control tests are performed on SKU rp367640?

Quality control typically includes SDS-PAGE purity analysis and protein concentration determination. Endotoxin testing may also be performed for cell-based application suitability. Exact QC data, including purity percentage, molecular weight confirmation, and endotoxin levels, are provided in the Certificate of Analysis accompanying each lot. Contact Aladdin Scientific for lot-specific documentation.

References

1. Ibba M, Söll D. Aminoacyl-tRNA synthesis. Annu Rev Biochem. 2000;69:617–650. doi:10.1146/annurev.biochem.69.1.617
2. Eriani G, Delarue M, Poch O, Gangloff J, Moras D. Partition of tRNA synthetases into two classes based on mutually exclusive sets of sequence motifs. Nature. 1990;347:203–206. doi:10.1038/347203a0
3. Aladdin Scientific. Recombinant Human FARSB Protein (rp367640) Product Datasheet. Aladdin Scientific; [date not specified].

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