Species: Mu
Applications: Func
Description
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.
