Journal: BMC Cancer

Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

doi: 10.1186/s12885-019-5388-8

Figure Lengend Snippet: Purification and Identification of Vimentin from DP-treated MDA-MB-231 cells. Proposed disulfide exchange reaction occurring between a cysteine sulfhydryl group on a target protein with ( a ) Z-ajoene (ZA) or its analogue ( b ) dansyl-ajoene (DP). ( c ) Lysate collected from MDA-MB-231 breast cancer cells treated with 25 μM ZA or DP show many dansyl-labelled proteins by immunoblot when probed with an anti-dansyl primary antibody in the DP-treated sample only. The experiment was performed under non-reducing conditions. ( d ) Separation of the dansyl-labelled proteins in MDA-MB-231 cell lysate by 2D gel electrophoresis under non-reducing conditions. A predominant band (circled) was observed in the immunoblot which was excised from the corresponding gel and identified by MALDI-TOF MS/MS to be vimentin

Article Snippet: The immunoblot was then run as described above and probed with an anti-dansyl primary antibody (1:7500, molecular probes).

Techniques: Purification, Western Blot, Two-Dimensional Gel Electrophoresis, Electrophoresis, Tandem Mass Spectroscopy

Journal: BMC Cancer

Article Title: The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells

doi: 10.1186/s12885-019-5388-8

Figure Lengend Snippet: Validation of Vimentin as an Ajoene Target. ( a ) Immunoblot of human recombinant vimentin treated with DP (100 μM) in the absence or presence of DTT (100 mM), probed with a primary anti-dansyl and anti-vimentin (H-84) antibody. ( b ) Human recombinant vimentin was treated with 100 μM ZA or DP and purified by SDS-PAGE. The band excised from the gel was digested with trypsin and fragments were identified by MS/MS MALDI-TOFF mass spectrometry. The Cys-328 containing fragment qvqsltcevdalk was detected in the control and treated samples carrying a 2+ charge where m/z = [M + 2H]2+. ( c ) In the samples treated with ZA or DP, the predicted m/z ratio of the modified fragment was observed

Article Snippet: The immunoblot was then run as described above and probed with an anti-dansyl primary antibody (1:7500, molecular probes).

Techniques: Biomarker Discovery, Western Blot, Recombinant, Purification, SDS Page, Tandem Mass Spectroscopy, Mass Spectrometry, Control, Modification