sCD40L Mouse, sf9

What is Mouse sCD40L produced in Sf9 cells?

Mouse sCD40L recombinant protein produced in Sf9 cells is a single, glycosylated polypeptide chain containing 155 amino acids (residues 112-260) with a molecular mass of 17.2kDa, though it migrates at 18-28kDa on SDS-PAGE under reducing conditions due to glycosylation. The protein is typically expressed with a 6 amino acid His tag at the C-Terminus and purified using proprietary chromatographic techniques to achieve greater than 95.0% purity as determined by SDS-PAGE . This recombinant form represents the soluble, extracellular domain of CD40L (also known as CD154), which is naturally a membrane glycoprotein expressed on the surface of T-cells. The recombinant version enables researchers to study CD40L functions without needing the membrane-bound form .

What are the biological functions of CD40L in the immune system?

CD40L (CD154) functions as a key immune regulator with multiple important biological activities. It stimulates B-cell proliferation and secretion of all immunoglobulin isotypes in the presence of cytokines, making it critical for humoral immune responses. Additionally, CD40L has been shown to induce cytokine production and tumoricidal activity in peripheral blood monocytes, demonstrating its role in innate immunity. In T-cell biology, it costimulates proliferation of activated T-cells, which is accompanied by the production of several important cytokines including IFN-gamma, TNF-alpha, and IL-2 . These multiple functions make CD40L an important molecule for studying various aspects of immune response coordination and cellular communication in research settings.

What are the alternative names for CD40L researchers should be aware of in literature searches?

When conducting literature searches on CD40L, researchers should be aware of numerous alternative designations to ensure comprehensive results. These include: CD40 ligand, T-cell antigen Gp39, TNF-related activation protein (TRAP), Tumor necrosis factor ligand superfamily member 5, CD154, CD40lg, CD40-L, Cd40l, gp39, HIGM1, IGM, IMD3, Ly-62, Ly62, T-BAM, and Tnfsf5 . Understanding these synonyms is particularly important when searching older literature or cross-referencing studies from different research groups that may use varying nomenclature for the same protein.

What are the optimal storage conditions for maintaining sCD40L Mouse, sf9 activity?

The optimal storage conditions for sCD40L Mouse protein depend on the intended usage timeframe. For short-term storage where the entire vial will be used within 2-4 weeks, the protein can be stored at 4°C. For longer periods of time, it should be stored frozen at -20°C . For long-term storage, it is strongly recommended to add a carrier protein such as 0.1% Human Serum Albumin (HSA) or Bovine Serum Albumin (BSA) to prevent protein adherence to surfaces and maintain stability . Multiple freeze-thaw cycles should be strictly avoided as they can significantly degrade protein activity and integrity. The formulation of the protein solution (0.25mg/ml) includes Phosphate Buffered Saline (pH 7.4) and 10% glycerol to help maintain stability during storage .

How should researchers prepare sCD40L Mouse, sf9 for experimental use?

When preparing sCD40L Mouse, sf9 for experimental use, researchers should follow a systematic approach to ensure optimal protein functionality. First, thaw the protein gently at 4°C or on ice rather than at room temperature to minimize protein degradation. After thawing, briefly centrifuge the vial to collect all liquid at the bottom before opening to prevent sample loss. For experiments requiring specific concentrations, dilute the protein using appropriate buffers that match the experimental conditions while maintaining protein stability (typically PBS with 0.1% BSA). When working with diluted protein, prepare only the amount needed for immediate use as diluted solutions have reduced stability compared to the stock solution. Furthermore, researchers should validate protein activity in their specific experimental system before proceeding with large-scale experiments, as activity can be influenced by experimental conditions or potential degradation during storage .

What quality control metrics should be assessed before using sCD40L Mouse, sf9 in experiments?

Before using sCD40L Mouse, sf9 in critical experiments, researchers should verify several quality control parameters to ensure reliable results. First, protein purity should be confirmed to be greater than 95% as determined by SDS-PAGE under reducing conditions, with expected migration between 18-28kDa . Biological activity testing is essential through functional assays that measure CD40L-specific effects, such as B-cell proliferation assays or cytokine induction in appropriate target cells. Researchers should also verify protein integrity by checking for signs of precipitation or aggregation by visual inspection and/or dynamic light scattering. For particularly sensitive applications, endotoxin level testing is recommended, especially for in vivo applications or primary cell culture experiments where endotoxin contamination could confound results. Finally, if extended storage has occurred, researchers may wish to re-verify protein concentration using quantitative methods such as Bradford or BCA assays prior to experimental use .

How can sCD40L Mouse, sf9 be utilized in immunological research models?

sCD40L Mouse, sf9 serves multiple experimental purposes in immunological research. Based on its biological functions, it can be used to stimulate B-cell proliferation and differentiation in vitro, making it valuable for studying humoral immune responses and antibody production mechanisms. The protein can also be employed to induce cytokine production in monocytes and macrophages, allowing researchers to investigate innate immune activation pathways . In T-cell research, sCD40L can be used as a costimulatory factor to enhance T-cell activation and study subsequent cytokine production (IFN-gamma, TNF-alpha, IL-2) . Additionally, search result indicates that sCD40L has been used in the context of chimeric SIV VLPs (virus-like particles), which significantly increased CD4+ and CD8+ T-cell responses to SIV Env, demonstrating its potential utility in vaccine research as an immunological adjuvant .

How can researchers verify the biological activity of sCD40L Mouse, sf9 in their experimental systems?

To verify the biological activity of sCD40L Mouse, sf9, researchers should employ functional assays that directly measure known CD40L-mediated responses. A primary verification method is to assess B-cell proliferation following sCD40L treatment, which can be measured using standard proliferation assays such as MTT/XTT, BrdU incorporation, or CFSE dilution. Researchers can also quantify immunoglobulin production from B-cells exposed to sCD40L using ELISA or ELISPOT techniques . For monocyte activation verification, measuring the induction of inflammatory cytokines (TNF-α, IL-6, IL-1β) by ELISA or qPCR provides evidence of functional activity. In T-cell systems, researchers can verify activity by measuring enhanced proliferation and cytokine production (particularly IFN-γ, TNF-α, and IL-2) when sCD40L is used as a costimulator alongside primary T-cell activation signals . Additionally, CD40 receptor engagement can be verified through analysis of downstream signaling events, such as NF-κB activation or upregulation of activation markers on target cells .

What are the methodological considerations when using sCD40L Mouse, sf9 with microvesicle delivery systems?

When using sCD40L Mouse, sf9 with microvesicle delivery systems, researchers must consider several methodological aspects based on similar approaches with membrane proteins. According to search result , Sf9 cells have been successfully used to produce microvesicles containing functional membrane transport proteins, suggesting similar approaches could be applied to CD40L. Researchers should consider the appropriate MOI (multiplicity of infection) when infecting Sf9 cells; an MOI of 1.0 has been reported effective for microvesicle production . The timing of microvesicle collection is crucial, with optimal harvesting typically occurring 3 days post-infection . Purification methods should involve careful centrifugation steps to separate microvesicles (~90 nm in diameter) followed by dialysis into appropriate buffers such as Ham's F12 media, and sterilization via 0.22 μm filters . For quantitation purposes, technologies like NanoSight can be employed to determine microvesicle concentration, with typical working concentrations around 10^10-10^11 microvesicles/ml . When delivering to target cells, researchers should verify successful incorporation through techniques like confocal immunofluorescence microscopy, possibly using fluorescently-tagged proteins to confirm delivery and localization .

How can sCD40L Mouse, sf9 be employed in vaccine development research?

sCD40L Mouse, sf9 shows promising applications in vaccine development research as indicated by search result . The protein can function as a molecular adjuvant to enhance immune responses to vaccine antigens. Research has demonstrated that chimeric virus-like particles (VLPs) containing CD40L significantly increased both CD4+ and CD8+ T-cell responses to viral antigens such as SIV Env . This suggests that incorporating sCD40L into vaccine formulations can enhance both helper T-cell and cytotoxic T-cell responses, potentially improving vaccine efficacy. For methodology, researchers can either incorporate CD40L directly into vaccine constructs (as demonstrated with VLPs) or administer it alongside vaccine antigens as a separate adjuvant. When producing CD40L for such applications, baculovirus expression systems in Sf9 cells have been successfully employed with specific primers and cloning strategies as detailed in the literature . Expression verification can be performed using flow cytometry with specific antibodies against CD40L. The MOI used for infection influences protein yield, with an MOI of 2 reported in the literature . Researchers should optimize transfection conditions using appropriate transfection kits (such as Baculo-Gold) and verify protein expression before advancing to vaccine formulation studies .

What are common challenges when working with sCD40L Mouse, sf9 and how can they be addressed?

Several technical challenges may arise when working with sCD40L Mouse, sf9. Protein stability issues are common, particularly with multiple freeze-thaw cycles which should be strictly avoided . Researchers can address this by aliquoting the protein upon first thaw and storing aliquots at -20°C. Loss of activity during storage can be mitigated by adding carrier proteins (0.1% HSA or BSA) as recommended for long-term storage . Protein adhesion to surfaces may occur, leading to reduced effective concentration; using low-binding tubes and pipette tips, and including carrier proteins in dilution buffers can minimize this issue. Variable cellular responses to sCD40L between experiments might occur due to differences in target cell activation states or receptor expression levels; researchers should standardize cell culture conditions and verify CD40 receptor expression on target cells where possible. If aggregation is observed, gentle centrifugation at low speed can help remove aggregates before use, though this may indicate compromised protein quality requiring fresh material. Finally, endotoxin contamination can confound results in sensitive assays; researchers should use endotoxin-free labware and consider endotoxin testing if unexpected inflammatory responses are observed .

How can researchers determine the optimal experimental conditions for sCD40L Mouse, sf9 in their specific assays?

To determine optimal experimental conditions for sCD40L Mouse, sf9 in specific assays, researchers should employ a systematic optimization approach. Begin with dose-response experiments testing a range of concentrations (typically 10-500 ng/ml) to identify the minimal effective concentration for the desired biological response . Time-course studies are essential to determine the optimal duration of treatment, as different CD40L-mediated effects may have distinct kinetics (e.g., early signaling events versus later gene expression changes). The buffer conditions should be optimized, considering that the protein is formulated in PBS with 10% glycerol ; researchers should test compatibility with their experimental buffers if dilution is significant. For cell-based assays, cell density and state of activation/differentiation can dramatically impact responsiveness to sCD40L and should be standardized. Co-stimulatory factors may be necessary for optimal activity in certain systems; for example, cytokines like IL-4 may enhance CD40L effects on B-cells. When designing experiments, appropriate positive controls (known CD40L responsive systems) and negative controls (CD40-blocking antibodies or CD40-negative cells) should be included to validate specific CD40L-mediated effects .

What analytical methods can be used to assess sCD40L Mouse, sf9 protein quality and integrity?

Multiple analytical methods can be employed to assess sCD40L Mouse, sf9 protein quality and integrity. SDS-PAGE under reducing conditions is the standard method to verify protein purity and molecular weight, with expected migration between 18-28kDa . Western blotting using anti-CD40L or anti-His tag antibodies provides confirmation of protein identity while assessing potential degradation products. Size exclusion chromatography (SEC) can evaluate protein aggregation state and homogeneity in solution. Dynamic light scattering (DLS) provides complementary information on protein size distribution and potential aggregation in native conditions. Mass spectrometry can verify protein sequence integrity and identify any post-translational modifications or unexpected variations. For glycosylation analysis, specialized techniques such as lectin blotting or glycan-specific staining can characterize the glycosylation pattern of the insect cell-produced protein. Circular dichroism (CD) spectroscopy can assess secondary structure integrity, particularly useful after storage or experimental manipulations. Most critically, functional assays measuring known biological activities of CD40L (as detailed in FAQ 3.3) provide the ultimate verification of biologically relevant protein integrity .

What is the amino acid sequence and key structural features of sCD40L Mouse, sf9?

The recombinant sCD40L Mouse protein produced in Sf9 cells has the following amino acid sequence and structural features:

Table 1: Amino Acid Sequence and Structural Features of sCD40L Mouse, sf9

The sequence represents the extracellular, soluble portion of mouse CD40L with a C-terminal 6× His tag added for purification purposes. The observed higher molecular weight on SDS-PAGE (18-28 kDa) compared to the calculated molecular weight (17.2 kDa) is likely due to glycosylation present on the protein when expressed in Sf9 insect cells .

What experimental models have demonstrated successful application of sCD40L Mouse, sf9?

Based on the search results and broader research applications of CD40L, the following table summarizes experimental models where sCD40L Mouse, sf9 has been successfully applied:

Table 2: Experimental Models Using sCD40L Mouse, sf9

These experimental models demonstrate the versatility of sCD40L Mouse, sf9 across various immunological research applications, from basic in vitro cell stimulation to more complex vaccine development studies .

How does the biological activity of different batches of sCD40L Mouse, sf9 compare in standardized assays?

While the search results don't provide specific batch-to-batch comparison data, researchers should employ standardized assays to assess consistency between different production lots of sCD40L Mouse, sf9. The following table outlines recommended standardized assays and their expected outcomes for batch comparison:

Table 3: Standardized Assays for Batch-to-Batch Comparison of sCD40L Mouse, sf9

For accurate batch-to-batch comparison, researchers should maintain reference standards of well-characterized lots with documented activity, and new batches should be tested in parallel with these reference standards. This approach ensures experimental reproducibility and reliable research outcomes when working with different production lots of sCD40L Mouse, sf9 .