Appearance ： Slightly yellow to pale yellowish brown powder or solid
Storage Condition： Store at 0-5oC
Shipping Condition： ambient temperature
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.
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
1) L. Makmura, M. Hamann, A. Areopagita, S. Furuta, A. Munoz and J. Momand. , "Development of a sensitive assay to detect reversibly oxidized protein cysteine sulfhydryl groups", Antioxid Redox Signal., 2001, 3, (6), 1105.
2) HH Wu, J.A. Thomas and J. Momand, "p53 protein oxidation in cultured cells in response to pyrrolidine dithiocarbamate: a novel method for relating the amount of p53 oxidation in vivo to the regulation of p53-responsive genes", Biochem J., 2000, 351, 87.
3) JR. Burgoyne, O. Oviosu and P. Eaton., "The PEG-switch assay: A fast semi-quantitative method to determine protein reversible cysteine oxidation", J Pharmacol Toxicol Methods., 2013, 68, (3), 297.
4) L. JTetsch, C. Koller, A. Donhofer and K. Jung, "Detection and function of an intramolecular disulfide bond in the pH-responsive CadC of Escherichia coli", BMC Microbiol., 2011, doi: 10.1186/1471-2180-11-74.
Analysis of the redox states of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase)
Figure 4 Detection of the redox state of the GAPDH
Analysis of the redox states of TRX (Thioredoxin) in HeLa cells
Figure 5 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 6 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)