Recombinant Bovine ORM1-like protein 2 (ORMDL2)

What is Recombinant Bovine ORM1-like Protein 2 (ORMDL2)?

Recombinant Bovine ORM1-like protein 2 (ORMDL2) is a full-length protein consisting of 153 amino acids that belongs to the ORMDL family of proteins. It is commonly produced with an N-terminal His-tag and expressed in E. coli expression systems for research purposes. The protein is identified in UniProt database with the accession number Q5E972 and functions as a regulatory subunit involved in various cellular processes .

How does Bovine ORMDL2 relate to other ORMDL isoforms?

Bovine ORMDL2 is one of three ORMDL isoforms (ORMDL1, ORMDL2, and ORMDL3) found in mammalian systems. These isoforms appear to have distinct but potentially overlapping physiological roles in regulating SPT (serine palmitoyltransferase) activity and other cellular processes. Recent research using CRISPR-Cas9 technology has generated cell lines expressing only single ORMDL isoforms, revealing their individual contributions to cellular functions .

What are the optimal storage and handling conditions for Recombinant Bovine ORMDL2?

Recombinant Bovine ORMDL2 is typically supplied as a lyophilized powder and should be stored at -20°C/-80°C upon receipt. For optimal stability:

• Aliquoting is necessary for multiple use to prevent repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

• The protein is typically stored in Tris/PBS-based buffer containing 6% Trehalose at pH 8.0

• Repeated freezing and thawing is strongly discouraged as it may compromise protein integrity

What is the recommended reconstitution protocol for lyophilized Bovine ORMDL2?

For proper reconstitution of lyophilized Bovine ORMDL2:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is typically recommended)

• Aliquot for long-term storage at -20°C/-80°C

This protocol helps maintain protein stability and prevents activity loss during storage periods .

How can I validate the functionality and purity of Recombinant Bovine ORMDL2?

Validation of Recombinant Bovine ORMDL2 can be accomplished through several methods:

• Purity Assessment: SDS-PAGE analysis, which typically shows >90% purity for commercial preparations

• Western Blotting: Using antibodies against ORMDL2 or the His-tag

• Functional Assays: Assessing SPT activity regulation, as ORMDL proteins are known to function as regulatory subunits

• Mass Spectrometry: For precise molecular weight confirmation

Note that antibodies available for ORMDL cannot typically distinguish between different ORMDL isoforms, so isoform-specific validation may require additional approaches such as siRNA knockdown followed by western blotting .

How has CRISPR-Cas9 been used to study ORMDL2 function?

CRISPR-Cas9 technology has been instrumental in elucidating ORMDL2 function through the following approaches:

• Generation of ORMDL double knockout and triple knockout cell lines

• Creation of cell lines expressing only one of the three ORMDL isoforms by deleting the other two

• Targeting genomic regions between exons 2 and 3 of each ORMDL isoform

• Validation through Sanger sequencing and protein expression analysis by western blotting

These genetic approaches have allowed researchers to determine the specific contributions of each ORMDL isoform to cellular processes and distinguish potentially redundant from unique functions .

What analytical techniques are used for detecting and quantifying ORMDL2 in biological samples?

Several analytical techniques are employed for ORMDL2 detection and quantification:

Researchers often combine multiple techniques to overcome the limitations of individual methods when studying ORMDL proteins .

What is known about ORMDL2's role in regulating serine palmitoyltransferase (SPT) activity?

ORMDL2, along with other ORMDL isoforms, functions as a regulatory subunit of serine palmitoyltransferase (SPT), the rate-limiting enzyme in sphingolipid biosynthesis. Recent research has focused on understanding the physiological importance of individual ORMDL isoforms in regulating SPT activity. Current findings suggest:

• ORMDL proteins may have partially redundant functions in SPT regulation

• Different ORMDL isoforms might have tissue-specific or condition-specific regulatory roles

• The precise mechanism of how ORMDL2 regulates SPT activity involves protein-protein interactions that can be studied using knockout cell lines

What are common challenges in working with recombinant ORMDL2 and how can they be addressed?

Researchers frequently encounter these challenges when working with recombinant ORMDL2:

• Protein Solubility Issues: ORMDL2 contains hydrophobic regions that may affect solubility

  • Solution: Optimize buffer conditions, consider detergent addition for membrane-associated proteins

• Antibody Cross-Reactivity: Antibodies may not distinguish between ORMDL isoforms

  • Solution: Use genetic approaches (knockout/knockdown) to validate isoform specificity

• Functional Assessment: Determining specific activity of recombinant ORMDL2

  • Solution: Develop in vitro SPT activity assays with purified components

How can researchers reliably quantify ORMDL2 in bovine samples?

For reliable quantification of ORMDL2 in bovine samples, researchers can employ:

• ELISA-based approaches: Similar to those developed for ORM1, using competitive ELISA techniques with polyclonal antibodies against the target protein

• Quantitative proteomics: TMT-6plex quantitative proteomics has been successfully used for bovine serum/plasma proteins and allows multiplexed analysis

• Western blot with calibration standards: Using purified recombinant ORMDL2 as a standard curve

When developing quantification methods, researchers should consider:

• Detection limits (typically in ng/mL range for most proteins)

• Linear range of detection

• Sample preparation requirements to minimize interference

What are emerging research areas involving ORMDL2 in bovine systems?

Emerging research involving bovine ORMDL2 includes:

• Comparative Functional Studies: Investigations comparing the functional roles of ORMDL1, ORMDL2, and ORMDL3 in bovine cells and tissues

• Biomarker Development: Exploration of ORMDL2 and related proteins (such as ORM1) as potential biomarkers for bovine diseases, following approaches similar to those used in proteomic serum profiling of Holstein Friesian cows

• Regulatory Networks: Characterization of the broader regulatory networks involving ORMDL2, including its potential interactions with other proteins and signaling pathways involved in lipid metabolism

• Physiological Significance: Determining the tissue-specific expression and function of ORMDL2 in bovine development and health

How do experimental approaches differ when studying ORMDL2 compared to other ORMDL isoforms?

The experimental approaches for studying ORMDL isoforms share methodological similarities but have important distinctions:

Researchers studying ORMDL2 specifically often need to employ genetic approaches to distinguish its functions from those of other ORMDL isoforms .

What statistical considerations are important when analyzing ORMDL2 experimental data?

When analyzing experimental data related to ORMDL2, researchers should consider:

• Appropriate Statistical Tests: For comparing two groups, an unpaired two-tailed Student's t-test is commonly used, with p ≤ 0.05 considered statistically significant

• Experimental Replication: All experiments should be performed independently at least three times to ensure reproducibility

• Controls: Include non-targeting controls when using genetic manipulation techniques such as CRISPR-Cas9 or siRNA

• Data Normalization: When quantifying ORMDL2 expression or activity, appropriate normalization to housekeeping genes or proteins is essential

• Multivariate Analysis: When studying ORMDL2 in relation to other biomarkers, multivariate statistical approaches may be necessary to account for interactions