Recombinant Human Ubiquitin+1

Recombinant Human Ubiquitin+1 is a valuable tool for research applications due to its unique properties and functional relevance in ubiquitin signaling pathways. Here are several reasons why you should consider using Recombinant Human Ubiquitin+1 in your research:

1. Functional Ubiquitin Variant: Ubiquitin+1 is a naturally occurring frameshift variant of ubiquitin that retains the ability to participate in ubiquitination processes. It can be conjugated to target proteins and incorporated into polyubiquitin chains, making it suitable for studying ubiquitin-dependent signaling and degradation pathways.

2. Model for Disease Mechanisms: Ubiquitin+1 is associated with certain pathological conditions, particularly polyglutamine diseases such as Huntington’s disease. At high levels, Ubiquitin+1 can overwhelm the proteasome system and inhibit its activity, which is thought to contribute to the accumulation of misfolded proteins and disease pathology. Using this variant allows researchers to model and study these disease mechanisms in vitro.

3. High Purity and Quality: Recombinant Human Ubiquitin+1 proteins, such as those produced by reputable suppliers, are typically over 95% pure and undergo rigorous quality control testing to ensure bioactivity. This ensures reproducible results in bioactivity assays and other experimental applications.

4. Versatile Applications: Recombinant Ubiquitin+1 can be used in a variety of assays, including:

   • In vitro ubiquitination assays to study E1, E2, and E3 enzyme activities.
   • Investigation of proteasome inhibition and its effects on protein degradation.
   • Analysis of ubiquitin chain formation and recognition by ubiquitin-binding domains (UBDs).
   • Studies on the impact of ubiquitin variants on cellular signaling and stress responses.

5. Convenience and Reliability: Recombinant Ubiquitin+1 is readily available and can be used as a positive control or as a specific tool to probe the effects of ubiquitin variants in your experimental system. Its consistent quality and activity make it a reliable reagent for both basic and applied research.

In summary, Recombinant Human Ubiquitin+1 is a powerful reagent for studying ubiquitin biology, disease mechanisms, and the functional consequences of ubiquitin variants in cellular processes.

Yes, recombinant human ubiquitin+1 can be used as a standard for quantification and calibration in ELISA assays. This is a well-established application in ubiquitin research.

Standard Preparation and Use

Recombinant ubiquitin+1 proteins are specifically formulated and validated for use as ELISA standards. When preparing your standard curve, the recombinant protein should be reconstituted according to manufacturer instructions, as these may be lot-specific. Typical standard curve ranges for ubiquitin quantification span from approximately 125 to 8000 pg/mL, though this can vary depending on your specific assay requirements.

Reconstitution Protocol

The reconstitution process typically involves dissolving lyophilized recombinant ubiquitin+1 in an appropriate assay diluent. For example, a common approach is to prepare an initial stock solution at 200 ng/mL by adding the diluent to the lyophilized standard vial, then serially diluting this to create your working standard curve. The recombinant protein is generally supplied in formulations that may include stabilizers such as bovine serum albumin (BSA), which is recommended for use in cell or tissue culture applications and as ELISA standards.

Important Considerations for Accuracy

When using recombinant proteins as ELISA standards, be aware that mass values calculated by ELISA may differ from those reported on the vial label. This discrepancy arises because large dilution steps are typically required to place the recombinant protein within the assay's linear range—often diluting from micrograms per milliliter to picograms per milliliter. Generally, a ±25% recovery of the stated amount is considered acceptable when using ELISA for protein concentration determination.

Validation

The recombinant ubiquitin+1 standard will recognize both natural and recombinant ubiquitin in your samples, making it suitable for quantifying ubiquitin across various sample types including serum, plasma, and cell culture supernatants.

Recombinant Human Ubiquitin+1 has been validated primarily for use in bioassays and ubiquitination assays in published research, particularly as a substrate or tool for studying ubiquitin-dependent processes in vitro and in cell-based systems.

Key validated applications in published research include:

• Bioassays: Used as a substrate in enzymatic assays to study ubiquitin ligases (E3s), deubiquitinases (DUBs), and ubiquitin-dependent signaling pathways.
• Ubiquitination assays: Employed to monitor the transfer of ubiquitin to target proteins, assess E1/E2/E3 enzyme activity, and characterize ubiquitin chain formation.
• Cellular assays: Utilized in cell or tissue culture systems to investigate ubiquitin-mediated protein modification, stability, and degradation.
• ELISA standard: Sometimes used as a standard in immunoassays to quantify ubiquitin or ubiquitinated proteins.

Published research examples demonstrate its use in:

• Cancer biology: Studying the role of ubiquitination in cancer cell signaling, protein degradation, and response to therapeutics.
• Protein quality control: Investigating the recognition and processing of misfolded proteins by the ubiquitin-proteasome system.
• Viral infection: Exploring ubiquitin-mediated regulation of viral proteins and host-pathogen interactions.
• Enzyme mechanism studies: Dissecting the specificity and activity of ubiquitin ligases and deubiquitinases.

In summary, Recombinant Human Ubiquitin+1 is a validated reagent for a broad range of biochemical and cell-based assays focused on ubiquitin-dependent processes, with applications spanning cancer research, protein homeostasis, and enzymology.

Reconstitution of Recombinant Human Ubiquitin+1 Protein

Recombinant Human Ubiquitin+1 is typically supplied in lyophilized form and requires proper reconstitution before use in cell culture experiments. The following guidelines outline the standard preparation procedures.

Reconstitution Protocol

Initial Reconstitution

Reconstitute the lyophilized protein by dissolving it in distilled water or an aqueous solution. A recommended starting concentration is 10 mg/mL. Allow the vial to reconstitute for 15-30 minutes at room temperature with gentle agitation. Avoid vigorous shaking, as this can cause foaming and protein denaturation.

Dilution and Stabilization

For long-term storage and to enhance protein stability, dilute the reconstituted protein with a carrier protein-containing solution such as 0.1% BSA, 10% FBS, or 5% HSA. Additionally, incorporate 5-50% glycerol (final concentration) to provide cryoprotection.

Concentration Optimization

For ubiquitination assays and cell culture applications, optimize the working concentration based on your specific experimental requirements. A recommended initial concentration range is 0.01-0.5 mM, though this should be adjusted according to your particular application protocol.

Storage Conditions

• Short-term storage: Store at 4°C for immediate use
• Long-term storage: Aliquot and store at -20°C to -80°C in a manual defrost freezer
• Critical: Avoid repeated freeze-thaw cycles, as these can compromise protein integrity and activity

Application Considerations

Ubiquitin+1 functions through formation of isopeptide bonds between its C-terminal glycine residue and lysine residues in target proteins. When designing your cell culture experiments, ensure your reaction conditions are optimized for your specific application, as ubiquitination efficiency can vary depending on the presence of appropriate E1, E2, and E3 enzymes.