MolBoolean Mouse/Rabbit Assay Kit

MolBoolean analysis of the interaction between E-cadherin (cat. NBP2-34475, magenta) and B-catenin (cat. NBP1-89989, green) in MCF7 cells, showing the relative quantification of free versus interacting protein fractions, indicated by the detection of rolling circle products (RCPs) in either one or two fluorescent channels. Data is normalized to total target protein levels (total RCPs).

MolBoolean analysis of the interaction between ACE2 (cat. NBP2-59036, magenta) and TMPRSS2 (cat. NBP2-38263, green) in human kidney, showing the relative quantification of free versus interacting protein fractions, indicated by the detection of rolling circle products (RCPs) in either one or two fluorescent channels. Data is normalized to total target protein levels (total RCPs).

• Reactivity: Mu
• Applications: Func

MolBoolean Mouse/Rabbit Assay Kit Summary

• Description: MolBoolean™ Mouse/Rabbit Assay Kit Summary
  
  

  The MolBoolean™ Mouse/Rabbit Assay Kit (Catalog # MolB00001) is a state-of-the-art tool for analyzing protein proximity within cells and tissues using advanced immunofluorescent detection. Specifically designed to quantify the levels of proteins A, B, and their interactions (AB), this kit works with user-selected mouse and rabbit primary antibodies and includes all necessary reagents for approximately 120 assays, following the provided protocol.

  Key Features of the MolBoolean™ Mouse/Rabbit Assay Kit

  • In Situ Protein Proximity Analysis: Allows detection of protein interactions directly within cells and tissues.
  • Simultaneous Detection: Accurately measures free and interacting fractions of proteins A and B, including their complex (AB).
  • Dual Fluorescent Reporters: Utilizes two immunofluorescent reporters with emission peaks at 590 nm and 664 nm for clear and distinct signals.
  • Versatile Primary Antibody Compatibility: Suitable for use with any mouse and rabbit primary antibodies chosen by the user.
  • Enhanced Signal Detection: Incorporates Rolling Circle Amplification (RCA) for amplified signal strength and clarity.
  • Standard Equipment Compatibility: Compatible with common immunofluorescent microscopy and equipment.

  7-Step Assay Process

  1. Proximity Probe Binding: Probes bind to the primary antibodies.
  2. Probe Arm Hybridization: Hybridization of probe arms to DNA circle oligos.
  3. DNA Nicking: Introduction of nicks in the DNA for further processing.
  4. Tag Oligo Incorporation: Incorporation of tagged oligonucleotides into the DNA.
  5. DNA Ligation: Ligation of the DNA ends.
  6. Rolling Circle Amplification: Amplification of the circular DNA to enhance signal.
  7. Detection: Visualization and analysis of the results.

  Kit Specifications

  Total Volume: 4.8 ml

  Assay Capacity: Sufficient for approximately 120 cell assays (40 µl/assay) and 60 tissue assays (80 µl/assay).

  The MolBoolean™ Mouse/Rabbit Assay Kit is an all-in-one solution for detailed and reliable protein interaction studies, offering exceptional precision and ease of use with standard laboratory equipment.
• Kit Type: Assay Kit

Applications/Dilutions

• Dilutions:
  • Proximity Ligation Assay
• Application Notes: Primary antibodies are not supplied as part of the MolBoolean kit. The user should include their own antibodies, one of rabbit and one of mouse origin, which have been validated in relevant applications.

Packaging, Storage & Formulations

• Storage: Store at -20C.

Notes

MolBoolean (TM) is a trademark of Atlas Antibodies AB. The product is for research use only and not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.

Background

Proteins are responsible for numerous biological processes within a cell including cell growth, signal transduction, and gene expression. Understanding protein-protein interaction levels can give insights into protein function and activation and is vital for therapeutic and drug development.



The MolBoolean assay is designed to detect protein interactions by utilizing anti-mouse and anti-rabbit secondary proximity probes and oligonucleotide setup. This assay facilitates simultaneous detection of both free and interacting (within ~40nm proximity) abundance levels of two proteins (i.e. protein A, protein B, and interaction proteins AB).



The principles of MolBoolean is similar to that of in situ proximity ligation assay (PLA) in that it utilizes proximity probes and rolling circle amplification (RCA), however it is unique in its use of a preformed DNA circle as an information receiver. The basic MolBoolean Assay steps include: proximity probe binding to target proteins followed by probe hybridization to the DNA circle, nicking and enzymatic digestion of the circle, reporter tag invasion, ligation of reporter tag to the circle, RCA resulting in long concatemeric products called RCPs, and RCP detection using fluorescently labeled tags. In terms of analysis, the single-labeled RCPs are indicative or free proteins, whereas dual-stained RCPs represent protein AB interactions. Quantification of free proteins or protein complexes can be done as number of RCPs per cell..



The MolBoolean assay is validated for use in both fixed cells and formalin fixed paraffin embedded (FFPE) tissue sections. This method is also effective in an array on organelles including the cell membrane, Golgi, ER, and mitochondria. The distinct output of RCPs gives the MolBoolean method greater signal intensity compared to traditional immunostaining, in addition to the ability to be quantified. Furthermore, an important feature of the MolBoolean assay is that it can distinguish RCPs generated through colocalization versus those from free proteins in close proximity. A benefit of being able to detect free and interacting proteins simultaneously is that this can be done in a single assay, saving both valuable resources and time.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Kits are guaranteed for 6 months from date of receipt.