Proteasome ELISA (Enzyme-Linked Immunosorbent Assay) Kits are analytical tools used to measure the levels and activity of proteasomes in biological samples. The proteasome is a crucial protease complex responsible for degrading ubiquitinated proteins, thereby regulating various cellular processes such as the cell cycle, signaling, and apoptosis. These kits are instrumental for studying proteasome function and dysfunction in diseases, including cancer, neurodegenerative disorders, and autoimmune diseases.

Key Components and Technical Details

Kit Components

• Coated Microplate: Typically, a 96-well plate is pre-coated with antibodies specific to proteasome subunits or proteasome activity markers.
• Standards: Known concentrations of proteasome subunits or activity markers to create a standard curve for quantification.
• Detection Antibody: An antibody specific to the proteasome or its subunits, often conjugated with an enzyme like horseradish peroxidase (HRP) for colorimetric detection.
• Substrate Solution: A reagent that reacts with the enzyme linked to the detection antibody to produce a colorimetric signal proportional to the proteasome amount or activity.
• Stop Solution: Usually sulfuric acid or hydrochloric acid, used to stop the enzyme reaction and stabilize the color for measurement.
• Washing Buffer: A buffer solution used to remove unbound proteins and reduce background noise.

Sample Preparation

• Cell Lysates/Tissue Homogenates: Samples should be properly prepared through homogenization and lysis to extract proteasome components or activity markers.
• Serum/Plasma: For blood-based assays, samples need appropriate handling to prevent proteasome degradation or activation.

Assay Procedure

• Coating: The microplate wells are pre-coated with antibodies specific to proteasome subunits or activity markers.
• Blocking: Non-specific binding sites are blocked to prevent false positives.
• Sample Incubation: Samples or standards are added to the wells, allowing for binding of proteasome components or markers.
• Washing: Excess and non-specifically bound substances are removed through washing.
• Detection: Detection antibodies are added to bind specifically to proteasome components or markers.
• Substrate Reaction: The substrate solution is added to produce a color reaction proportional to the proteasome level or activity.
• Stop Reaction: A stop solution is added to halt the color development.
• Reading: The optical density is measured using a microplate reader at the appropriate wavelength (usually 450 nm).

Quantification

• The standard curve created from known concentrations of proteasome standards is used to determine the concentration or activity of proteasome in the samples based on optical density readings.

Applications

• Disease Research: Studying proteasome involvement in diseases such as cancer and neurodegenerative disorders.
• Drug Development: Evaluating the effect of potential therapeutic agents on proteasome activity.
• Cell Biology: Investigating the role of the proteasome in various cellular processes and stress responses

Proteasome ELISA Kits are valuable tools for researchers and clinicians, offering precise measurement of proteasome levels and activity, which is essential for understanding proteasome function and its implications in health and disease.