MolBoolean™ is a kit for in situ protein proximity analysis in tissue and cells developed by Atlas Antibodies. Unlike traditional methods, it provides spatial quantitative analysis of protein-protein interactions by the simultaneous detection of free and interacting proteins (~40 nm proximity). 

The MolBoolean™ assay applies a Boolean logic at a molecular level to distinguish between free and interacting proteins, mapping protein interactions with high spatial specificity.

The OR logic identifies either protein A or B independently, signaling that they are present but not necessarily interacting.
The AND logic is applied when proteins A and B are interacting; they are detected together as complex AB, indicating a combined "true" interaction.

Comparative table that highlights the advantages of MolBoolean™ over in situ PLA and FRET for studying protein-protein dynamics:

MolBoolean provides spatial quantitative analysis of protein-protein interactions by the simultaneous detection of free
and interacting proteins. MolBoolean™ offers significant benefits in fields requiring complex spatial interaction and quantification data such as:

• Dual Detection Capabilities: MolBoolean detects both interacting and non-interacting endogenous fractions for two protein targets in cells and tissues. The more comprehensive detection capability increases the spatial information that can be extracted from the experiment, allowing researcher to determine locations of positive or negative protein interactions.
• Data normalization: with MolBoolean, the number of protein interactions can be normalized to the total number of target proteins. This is crucial because target protein levels can be influenced by various factors such as cell treatments or disease states. By providing a normalized measure, MolBoolean™ allows for more accurate comparisons between samples.
  
• High Sensitivity
  Achieve 1000-fold amplification of signal for exceptional clarity in detecting interactions, allowing visualization and quantification of low abundant proteins. The MolBoolean technology benefits from a built-in signal amplification mechanism, allowing it to detect individual molecules with high sensitivity. This makes it not only versatile but also accessible for detecting small amounts of free proteins that might otherwise go unnoticed, giving valuable insight into the dynamic balance of protein interactions.
• Consistent molecular process: The rolling circle products (RCPs) stemming from free or interacting proteins are generated through the same molecular process steps. This ensures uniform signal efficiencies, reducing uncertainty during data analysis.
• Compatibility
  Easily integrate MolBoolean™ with your existing, validated antibodies and protocols, offering flexibility and saving time in diverse research applications.

MolBoolean™ Mouse/Rabbit is a kit for in situ protein proximity analysis in tissue and cells. The MolBoolean™ assay utilizes a proprietary oligonucleotide setup that enables the simultaneous detection of both free and interacting (~40 nm proximity) fractions for two proteins of interest (protein A, protein B and interaction proteins AB). It uses two different immunofluorescent detection reporters with maximum emission wavelengths at 590 nm (ATTO565, TxRed filter or similar) and at 664 nm (ATTO647N, Y5 filter or similar), respectively.

MolBoolean™ Mouse/Rabbit can be performed with the user’s primary antibodies of choice (raised in mouse and rabbit). MolBoolean™ relies on anti-mouse and anti-rabbit secondary proximity probes and rolling circle amplification (RCA) as a mean to amplify signal.

A series of molecular steps, performed in the MolBoolean™ assay, incorporates information into the amplified products indicating antibody target engagement of individual versus interacting proteins. This information is converted to fluorescent signals by the binding of detection reporters, allowing visualization on a conventional fluorescent microscope. An image analysis software is provided to segment and differentiate fluorescent signals, enabling the relative quantification of free versus interacting fractions for the two analyzed protein targets in the sample.

The MolBoolean™ assay follows a 2-days 7-steps workflow to determine if target proteins are interacting (AB) or present individually (A, B).

Day 1 (2 hours): blocking and primary antibodies incubation.
Day 2 (7-8 hours): probe binding, tagging, amplification, and detection.

(STEP 1) Proximity probes bind to primary antibodies attached to the proteins.
(STEP 2) Proximity probes link to the DNA circle.
(STEP 3) DNA nicking prepares the DNA for tagging.
(STEP 4) Tag oligos are added to mark the proteins.
(STEP 5) DNA ligation connects the DNA segments.
(STEP 6) Rolling circle amplification (RCA) boosts the signal for easier detection.
(STEP 7) Detection reveals both interacting and separate proteins.

The MolBoolean™ kit includes 15 separate tubes: enzymes (nickase, ligase, polymerase), buffers, oligos for tagging and detection, secondary antibody probes and additional reagents such as blocker, and diluent.

1:Blocker (4x) - 2:Diluent (1x) - 3:Probe A (80x) - 4:Probe B (80x) - 5:Circle (40x) - 6:Additive (40x) - 7:Buffer A (10x) - 8:Nickase enzyme (80x) - 9:Buffer B (8x) - 10:Tag oligos (40x) - 11:Ligase enzyme (80x) - 12:Polymerase enzyme (40x) - 13:Buffer C (5x) - 14:Detection oligos (40x) - 15:Buffer D (10x).

The total volume of 4.8 ml, is sufficient for approx. 120 assays in cells (40 μl/assay) and 60 assays in tissue (80 μl/assay).

MolBoolean is a kit that can be used with the customer’s choice of primary antibodies (mouse /rabbit). MolBoolean is compatible with protocols already established in the lab for primary antibodies. This flexibility allows researchers to seamlessly integrate MolBoolean into their existing workflows without requiring the purchase of specialized antibodies, making it a convenient and adaptable tool for studying protein interactions and free protein levels.

Be aware that the specificity of the primary antibodies can affect the accuracy of MolBoolean results. It is important to validate the primary antibodies to ensure that they specifically target the proteins of interest before applying the MolBoolean technology. We recommend the use of Enhanced validated primary antibodies. 

Rivas-Santisteban R, et al, (2024) GPR88 impairs the signaling of kappa opioid receptors in a heterologous system and in primary striatal neurons. Neuropharmacology. 2024 Nov 27:110242. doi: 10.1016/j.neuropharm.2024.110242. Epub ahead of print. PMID: 39613254.

Kotliar IB, et al, (2024) Multiplexed mapping of the interactome of GPCRs with receptor activity-modifying proteins. Sci Adv. 2024 Aug 2;10(31):eado9959. Epub 2024 Jul 31. PMID: 39083597; PMCID: PMC11290489.

Malmqvist M, et al, (2024) Quantifying dopamine D2 and adenosine A2A receptor interactions in rat brain using MolBoolean™ technology. #8217 Society for Neuroscience, SfN, (2024).

Raykova D, et al, (2023) A method for Boolean analysis of protein interactions at a molecular level. Nat Commun. 2023 Sep 6;14(1):5450. doi: 10.1038/s41467-023-41325-3. Erratum for: Nat Commun. 2022 Aug 13;13(1):4755. doi: 10.1038/s41467-022-32395-w. PMID: 37673885; PMCID: PMC10482831.

Rivas-Santisteban R, et al, (2023) Boolean analysis shows a high proportion of dopamine D2 receptors interacting with adenosine A2A receptors in striatal medium spiny neurons of mouse and non-human primate models of Parkinson’s disease. Neurobiol Dis. 2023 Nov;188:106341.doi: 10.1016/j.nbd.2023.106341. Epub 2023 Oct 31. PMID: 37918757.

Prof. Ola Söderberg, Uppsala University, Sweden (inventor of the MolBoolean technology) "Over the last two decades we have developed several methods to visualize proximity between proteins, MolBoolean being the latest and most advanced method.  In contrast to PLA, it provides the ability to simultaneously monitor both the levels of free proteins and their interactions in fixed cells and tissue. The MolBoolean method was designed to incorporate targeting of both free and interacting proteins with one unified approach, instead of relying on orthogonal methods, keeping the efficiency in their reporting more similar." 

Dr. Ilana Kotliar and Dr. Yuanhuang Chen, Prof. Thomas Sakmar, Laboratory at Rockefeller University, New York, USA "The MolBoolean fluorescence in situ protein proximity detection system exceeded our expectations as a robust analytical method to detect protein-protein interactions in cell membranes.  With validated antibodies, the system could quantitate both individual components and complexes. Benchmarking and workflow were straightforward. We used chamber slides and the CellProfiler imaging and data processing pipelines. The results are spectacular. "

Dr. Inés Rodríguez González and Prof. Dr. Ralph Kehlenbach, Georg-August-University, Göttingen, Germany "MolBoolean analysis is a very interesting approach to obtain quantitative data of in situ protein interactions that are probably very weak or transient and difficult to capture by conventional biochemical methods. The biggest advantage of MolBoolean is that, in addition to detecting two proteins in close proximity, it allows the quantification of the individual pools of protein 1 and 2, facilitating a direct comparison with the interacting fraction. The MolBoolean protocol is also informative and easy to follow. Deconvolution of microscopy images to further increase their resolution and the analysis of the obtained RCPs can be performed in open access software such as FIJI and Cell Profiler. All in all, we believe that MolBoolean analysis is a technology with many advantages and has the potential to be exploited and applied in different areas of cell biology.”

Prof. Rafael Franco, Network Center for Biomedical Research in Neurodegenerative Diseases, University of Barcelona, Spain. "Since the hypothesis of GPCR heteromers at the cell surface there has been much advance, except in what concerns the relative numbers of interacting versus non-interacting receptors. When we knew of “MolBolean” we wanted to test it and we are surely happy with the experience, and with the results. We used the MolBoolean kit to study the expression levels of A2A (A2AR)/dopamine D2 (D2R) receptor heterodimers in the striatum of rat and non-human primate models of Parkinson’s disease. MolBoolean allowed us to quantify the levels of receptor heterodimers. More importantly, the ability to also detect non-interacting receptors enabled us to determine the percentage of total receptors that are forming these heterodimers, shedding more light on how A2AR mediate their antagonistic regulation on motor control in the brain. MolBoolean workflow and protocol were straightforward and easy to follow.

Instructions For Use

Product Data Sheet 

Product Safety Data Sheet

Parallel Spectral Deconvolution Plugin

Diffraction PSF 3D Plugin

Image Analysis Pipeline