Proteomic Analysis of EV-Treated H69 Cells

H69 cells were cocultured with the quantity of EVs that corresponded to 1.2 µg/mL of ES protein in PBS for 30 minutes, 3 hours, and 16 hours. Cells were washed using PBS containing protease inhibitor cocktail and lysed in 5 M urea, 2 M thiourea, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X–100, and 40 mM Tris (pH 7.4). Each sample was ground with a TissueLyser II (Qiagen) and centrifuged at 12 000g for 20 minutes. The protein supernatant was precipitated with cold methanol and centrifuged at 8000g. Air-dried pellets were redissolved in 0.5 M triethylammonium bicarbonate (TEAB)/0.05% SDS and then centrifuged at 12 000g. Samples were resuspended in 0.5 M TEAB prior to reduction, alkylation, digestion, and iTRAQ labeling (AB Sciex) and then were analyzed on a 5600 TripleTOF mass spectrometer as described previously [24 (link)].

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Chaiyadet S., Sotillo J., Smout M., Cantacessi C., Jones M.K., Johnson M.S., Turnbull L., Whitchurch C.B., Potriquet J., Laohaviroj M., Mulvenna J., Brindley P.J., Bethony J.M., Laha T., Sripa B, & Loukas A. (2015). Carcinogenic Liver Fluke Secretes Extracellular Vesicles That Promote Cholangiocytes to Adopt a Tumorigenic Phenotype. The Journal of Infectious Diseases, 212(10), 1636-1645.

Publication 2015

TissueLyser II iTRAQ labeling 5600 TripleTOF mass spectrometer

2 thiourea Alkylation Cells Cold Digestion Methanol Ml 1 protein Pellets Protease inhibitor Protein Sodium dodecyl sulfate Triethylammonium bicarbonate Tris Triton x 100 Urea

Corresponding Organization : Australian Institute of Tropical Health and Medicine

Other organizations : Graduate School USA, University of Cambridge, University of Queensland, QIMR Berghofer Medical Research Institute, University of Technology Sydney, George Washington University, Khon Kaen University

Top 5 similar protocols

Variable analysis

independent variables

Incubation time of H69 cells with extracellular vesicles (EVs)

dependent variables

Protein expression in H69 cells

control variables

Quantity of EVs corresponding to 1.2 µg/mL of ES protein in PBS
Lysis buffer composition (5 M urea, 2 M thiourea, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X–100, and 40 mM Tris (pH 7.4))
Protein precipitation and resuspension in 0.5 M triethylammonium bicarbonate (TEAB)/0.05% SDS
Reduction, alkylation, digestion, and iTRAQ labeling of samples
Analysis on a 5600 TripleTOF mass spectrometer

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!