Unlock protein-protein interaction secrets
in cells and tissue with
MolBoolean™

New from Atlas Antibodies, Molboolean™ in situ protein proximity assay goes beyond proximity ligation.

Featuring innovative technology developed in collaboration with Professor Ola Soderberg (Uppsala University)

Get the full picture. MolBoolean enables simultaneous imaging of the interacting fraction between two protein targets (AB) AND the non-interacting pools of each free protein (A pool, B pool).

Background

Current popular methods for detecting interaction between two target proteins in cells or tissue, notably proximity ligation assays (PLA), are effective but do not provide information on the amount of non-interacting targets. This is critical because target protein levels can be influenced by factors such as cell treatment or disease state. Molboolean goes beyond proximity ligation by providing spatial and quantitative information not only on the interacting target population, but also on the non-interacting fractions of each target. With this information the fraction of interacting protein in cells can be normalized to the total amount of target proteind, allowing for more accurate comparison between samples and thus, more confident conclusions.

MolBoolean™ Assay Mouse/Rabbit  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).

A MolBoolean™ Mouse/Rabbit assay is performed with two primary antibodies, one for each target. One primary antibody must be of mouse origin, the other of rabbit origin. Proprietary oligonucleoditde-conjugated anti-mouse and anti-rabbit secondary antibodies are used to enable rolling circle amplification (RCA) to amplify signals.

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.

MolBoolean™ Features:

• Complete spatial quantitative analysis of protein-protein interactions by simultaneous detection of free and interacting proteins.
• Accurate quantification by normalization of interaction data to total target protein levels.
• Biologically relevant data without the need for engineered protein expression.
• 1000-fold increased fluorescence signal by Rolling Circle Amplification, allowing detection and quantification of low abundant proteins.
• Adaptable to different research needs. Universal kit that can be used with the customer’s choice of primary antibodies.
• Validated in both cells and tissue.
• EXPLORE A NEW FRONTIER OF PROTEIN-PROTEIN INTERACTION! 

Experimental Example

MolBoolean™ protein proximity data normalization to account for dynamic changes in protein complex formation treatment of HaCAT cells following TGF-β1 treatment. To account for the increased cell area and consequent increase in the total number of rolling circle products (RCP) per cell detected in the treated condition, all MolBoolean™ data was normalized by to the total number of RCPs detected in cells. A significant decrease was observed in the fraction of E-cadherin–βcatenin complexes as a result of the increase in free E-cadherin proteins.
See Moolbolean™ Application Note, Figure 3 for full details and comparative results obtained by PLA.

 

Molboolean Image Aquisition and Analysis

Quick summary of MolBoolean imaging parameters
Imaging	confocal (preferred) or widefield fluorescent microscope.
Filters/Detectors	matching ATTO565 (for A protein) 576-620 ex/em matching ATTO647N (for B protein) 657-750 ex/em DAPI filter if nuclei are counterstained
Image Acquisition	as close as possible to Nyquist rate with 63x or 100x objective https://svi.nl/Nyquist-Calculator
Image Processing	e.g. Huygens deconvolution (commercial) e.g. ImageJ + plugins (freeware) ImageJ plugin: “Parallel Spectral Deconvolution” plugin ImageJ plugin: “Diffraction PSF 3D” plugin
Image Analysis Software	CellProfiler software CellProfiler pipeline for MolBoolean image analysis

Product Description

Molboolean Kit Contents
tube #	Contents	tube #	Contents
1	Blocker (4x)	9	Buffer B (8x)
2	Diluent (1x)	10	Tag Oligos (40x)
3	Probe A (80x)	11	Lgiase enzyme (80x)
4	Probe B (80x)	12	Polymerase Enzyme (40x)
5	Circle (40x)	13	Buffer C (5x)
6	Additive (40x)	14	Detection Oligos (40x)
7	Buffer A (10x)	15	Buffer D (10x)
8	Nickase Enzyme (80x)

Molboolean Resources

Video: Introduction to Molboolean (~4 min)

Webinar: "Molboolean: A Brand New Method For The Analysis Of Protein Interactions in Cells and Tissue".

Signup to watch at Atlas Antibodies website.

Video: MolBoolean Image Analysis Video Tutorial

      

Product List

Molboolean™ Ordering Information

Now for a limited time, save big on Atlas Antibodies for use with Molboolean. 50% off Cosmo Bio USA's list price. Qualifying antibodies include Atlas PrecisA monoclonals and Atlas Antibodies to markers found in the Interaction Atlas on the HPA website. Limit 10 Vials per customer. Must be purchased together with a Molboolean kit. Orders must mention ATLAS-MOLBOOLEAN to receive discounted price. 

 

Product name	Storage	Catalog Number
MolBoolean Mouse/Rabbit Kit (sufficient for ~60 assays with tissue, ~120 with cells) Primary anti-target A antibody and anti-target B antibody (one mouse and one rabbit) not included.	-20C	ATL-MolB00001