The hippocampal lysates were processed for proteome analysis, as described previously (33). For digestion, 15 μl (~100 μg) of homogenate was diluted to 160 μl of ammonium bicarbonate in LoBind tubes. The proteins were denatured using 10 μl of 1% (w/v) RapiGestTM SF surfactant (Waters, Manchester, UK) in 25 mM ammonium bicarbonate followed by incubation at 80°C for 10 min. Samples were reduced by adding 10 μl of 60 mM dithiothreitol and incubated at 60°C for 10 min and alkylated by adding 10 μl of 180 mM iodoacetamide and incubated at room temperature in the dark for 30 min. Trypsin (Sigma, UK) was reconstituted in 50 mM acetic acid to a concentration of 0.2 μg/μl. Digestion was performed by the addition of 10 μl of trypsin to the samples followed by incubation at 37°C overnight. Trifluoroacetic acid (TFA) was added to each sample for acidification, and the samples were incubated at 37°C for 45 min. Samples were centrifuged at 17,000 × g for 45 min, and the supernatant was transferred to a LoBind tube. The centrifugation step was repeated, and 10 μl of supernatant was transferred to a total recovery vial for LC-MS analysis. Pre- and post-acidification digest was analyzed by SDS-PAGE to confirm complete digestion.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.