Modification of protein thiols is one of the most important post-translational modifications and it occurs according to the redox states in cells. It has recently been revealed that the modifications of thiol groups control cellular functions such as transcription, protein expression, cell death, etc. Therefore, detection of the redox states of the individual protein is important to understand cellular events. -SulfoBiotics- PEG-PCMal is a reagent to visualize the redox states of protein by electrophoretic analysis. PEG-PCMal has a maleimide group that can bind covalently to a protein thiol group. A mobility shift corresponding to about 5 kDa is observed by the electrophoretic analysis when one molecule of PEG-PCMal binds to a thiol group of the target protein. Thus, the number of free thiol groups on a protein can be clearly identified by SDS-PAGE through the mobility shift assay. The conventional reagents, PEG-Maleimide, have been widely used in mobility shift assays, however, transfer efficiency and antibody recognition on western blot are low due to the PEG chains labeled in protein. Because this reagent has a UV photocleavable moiety in the molecule, the PEG chains are cut off from the labeled protein in the gel with UV irradiation after the electrophoresis. Therefore, the protein treated with UV irradiation can be transferred from the gel to PVDF membrane and detected by antibodies.
Figure 1 Schematic protocol of PEG-PCMal
Figure 2 Structure of PEG-PCMal
Figure 3 Gel shift assay mechanism by PEG-PCMal depend on protein redox state
Analysis of the redox states of TRX (Thioredoxin) in HeLa cells
Figure 4 Visualization of the redox state of the TRX in HeLa cells
Analysis of the redox states of protein (ATP synthase γ subunit) in Arabidopsis thaliana
Figure 5 Visualization of the redox state of the photoresponsive protein in Arabidopsis thaliana
Dr. Toru Hisabori (Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology)
Dr. Keisuke Yoshida (Laboratory for Chemistry and Life Science, Institute of Innovative Research, Tokyo Institute of Technology)
Dr. Satoshi Hara (School of Life Science and Technology, Tokyo Institute of Technology)
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