Protein Retention Expansion Microscopy Anchoring Proteins to Gel
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Solution Overview
Problem
Expansion microscopy (ExM) faces challenges in retaining native proteins within samples and requires custom-made reagents, limiting its adoption due to the need for gel-anchorable fluorophores and inability to image genetically encoded fluorophores without antibody labeling.
Innovation Solution
The protein retention ExM (proExM) method anchors proteins to a swellable gel using a bifunctional crosslinker, allowing for nanoscale imaging of immunostained cells and tissues, even after nonspecific proteolytic digestion, and enabling the use of commercially available secondary antibodies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If proteins are not anchored to the gel, then the gel remains swellable and expandable, but proteins are lost during proteolytic digestion
Solution Approach 1:
A bifunctional crosslinker serves as an intermediary molecule that connects proteins to the gel matrix. The crosslinker has one functional group that reacts with protein amino groups and another that reacts with gel functional groups, thereby anchoring proteins to the gel without requiring custom-made reagents or complex procedures
Solution Approach 2:
The method changes the chemical state of proteins by introducing crosslinker molecules that form covalent bonds with protein amino groups. This parameter change (from unanchored to crosslinked) enables proteins to withstand proteolytic digestion while remaining attached to the expanding gel matrix
2Measurement precision
If custom-made gel-anchorable fluorophores are used, then protein labeling is achieved, but the barrier for researcher adoption increases
Solution Approach 1:
The bifunctional crosslinker serves multiple functions: it acts as both a gel anchoring agent and a protein labeling reagent. By using commercially available crosslinkers with appropriate functional groups, the method eliminates the need for custom-made gel-anchorable fluorophores while maintaining precise protein labeling capability
Solution Approach 2:
The method replaces expensive, custom-synthesized gel-anchorable fluorophores with inexpensive, commercially available bifunctional crosslinkers. These standard reagents can be purchased off-the-shelf, dramatically reducing the barrier for researcher adoption while achieving the same labeling function
3Stability of the object's composition
If nonspecific protease is used for homogenization, then mechanical properties are homogenized, but proteins are digested and lost
Solution Approach 1:
The crosslinking step is performed preliminarily before proteolytic digestion to protect proteins from being digested. By anchoring proteins to the gel matrix in advance, the method prevents protein loss during the homogenization process while still allowing the protease to homogenize the mechanical properties of the specimen
4Ease of operation
If genetically encoded fluorophores are used without antibody labeling, then direct imaging is enabled, but they cannot be imaged in original ExM
Solution Approach 1:
The bifunctional crosslinker serves as an intermediary that directly anchors genetically encoded fluorophores to the gel matrix. This eliminates the need for antibody labeling while preserving the fluorophore signal through the proteolytic digestion process, enabling direct imaging of genetically encoded markers
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
proExM preserves fluorescent signals from genetically encoded proteins and conventional antibodies, achieving high retention rates and enabling super-resolution imaging with minimal distortion, thus overcoming the limitations of the original ExM protocol.
Implementation Method 1
conjugating proteins within the sample with a bifunctional crosslinker, wherein the bifunctional crosslinker comprises a protein-reactive chemical group and a swellable material-reactive chemical group
Implementation Method 2
swelling the swellable material to form an expanded sample
Data Source
Figure 1
Figure 2
Figure 3A~3C
AI summary
The invention provides a method termed protein retention ExM (proExM), in which proteins, rather than labels, are anchored to the swellable gel, using a cross-linking molecule. This proExM strategy can be used to perform nanoscale imaging of immunostained cells and tissues as well as samples expressing various FPs as fluorescent signals from genetically encoded fluorescent proteins and/or conventional fluorescently labeled secondary antibodies and streptavidin that are directly anchored to the gel are preserved even when subjected to the nonspecific proteolytic digestion.