The wild-type type-I AFP was purchased from A/F Protein Inc. (Waltham, MA, USA) (99.0%). The cysteine-substituted HPLC6 peptides were synthesized by Biomatik Corporation (Wilmington, DE, USA). Their purities are > 95%. The MSL (4-maleimido-2,2,6,6-tetramethyl-1-piperidinyloxy) spin label was purchased from Sigma-Aldrich (CAS Number 15178–63-9).
A buffer solution (pH = 7.4) was prepared with Phosphate buffered saline tablets (Cas # BP2944–100) purchased from Fisher scientific. One (1.0) mg of cysteine-substituted HPLC6 peptides were dissolved in 0.5 ml of the buffer solution in an Eppendorf vial (2-ml volume), and 1.565 mg of MSL were dissolved in 0.25 ml of ethanol. The two solutions were then mixed together in a closed vial wrapped with aluminum foil. The reaction took place under shaking condition via a vortex mixer for 2–4 h in a cold room kept at 4 °C. After reaction, the sample was lyophilized. Then, the solid powder was dissolved in 0.5 ml of deionized (DI) water and transferred into a dialysis tube with a membrane of 1 kDa mesh size. The dialysis tube was purchased from Sigma-Aldrich (CAS Number PURD10005–1KT). The dialysis was done in DI water at 4 °C. Several changes of water were made, at 1, 2, 4 h, …, and overnight. The dialyses were conducted until no EPR signal could be detected in the DI water. The purified products were lyophilized and stored in a − 20 °C freezer.
A MALDI-TOF mass spectrometer (Voyager-DE™ STR Biospectrometry™ Workstation) was used to verify the masses of the spin-labeled HPLC6 mutants. The matrix was comprised of alpha-cyano-4-hydroxycinnamic acid, 3% TFA, acetonitrile, and DI water. The mass spectra of the original cysteine-substituted HPLC6 mutants and their spin-labeled versions are provided in thesupplemental file .
A buffer solution (pH = 7.4) was prepared with Phosphate buffered saline tablets (Cas # BP2944–100) purchased from Fisher scientific. One (1.0) mg of cysteine-substituted HPLC6 peptides were dissolved in 0.5 ml of the buffer solution in an Eppendorf vial (2-ml volume), and 1.565 mg of MSL were dissolved in 0.25 ml of ethanol. The two solutions were then mixed together in a closed vial wrapped with aluminum foil. The reaction took place under shaking condition via a vortex mixer for 2–4 h in a cold room kept at 4 °C. After reaction, the sample was lyophilized. Then, the solid powder was dissolved in 0.5 ml of deionized (DI) water and transferred into a dialysis tube with a membrane of 1 kDa mesh size. The dialysis tube was purchased from Sigma-Aldrich (CAS Number PURD10005–1KT). The dialysis was done in DI water at 4 °C. Several changes of water were made, at 1, 2, 4 h, …, and overnight. The dialyses were conducted until no EPR signal could be detected in the DI water. The purified products were lyophilized and stored in a − 20 °C freezer.
A MALDI-TOF mass spectrometer (Voyager-DE™ STR Biospectrometry™ Workstation) was used to verify the masses of the spin-labeled HPLC6 mutants. The matrix was comprised of alpha-cyano-4-hydroxycinnamic acid, 3% TFA, acetonitrile, and DI water. The mass spectra of the original cysteine-substituted HPLC6 mutants and their spin-labeled versions are provided in the
