Read e-book online Activity-Based Proteomics: Methods and Protocols PDF
By Herman S. Overkleeft, Bogdan I. Florea
This quantity specializes in explorative activity-based proteomics,biomedical purposes of activity-based proteomics, and chemical ideas in activity-based proteomics delivering a concise assessment of activity-based protein profiling. Written within the hugely winning Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, with ease reproducible laboratory protocols, and pointers on troubleshooting and heading off recognized pitfalls.
Authoritative and state-of-the-art, Activity-Based Proteomics: tools and Protocols goals to make sure winning leads to the additional research of this very important field.
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Extra info for Activity-Based Proteomics: Methods and Protocols
Repeat this step once more. 7. If you plan to use the Typhoon scanner to detect your proteins by fluorescence, make sure you use a fluorescent protein ladder. This facilitates detection of your proteins. Fluorescent protein markers are usually quite expensive. Many of the regular protein ladders that contain red marker bands are fluorescent under the settings used to detect rhodamine-labeled proteins. The blue marker bands are often fluorescent when using the settings for Cy5. We usually use Spectra Multicolour Broad Range Protein Ladder from Thermo (Cat.
Repeat the washes with the washing solution (see steps 11 and 12) four more times. 14. 5 mL Eppendorf tubes with “A” and “B”. 15. Remove the last wash from the Falcon tubes. In each tube should be around 1 mL liquid and beads. Disperse the beads by pipetting up and down with a blue, cut-off pipette tip. Quickly transfer the beads to a fresh Eppendorf tube. Wash the Falcon tube with 500 μL 1× PBS and transfer the solution to the appropriate Eppendorf tube. 16. Collect the beads by centrifugation (16,000 × g, 1 min, 15 °C) and remove the supernatant carefully.
44. After each washing step the gel slices will become more opaque/white as acetonitrile extracts water from the gel slices. The remaining substance is solid polyacrylamide with your proteins trapped in the matrix. 45. When you pipet ACN you will notice that quickly a droplet will form at the end of the tip. 3 kPa H2O). It evaporates in the pipette (expansion) and presses the liquid out of the pipet. When you pipette ACN, be quick. Take care that you do not drop any ACN on the labels of your Eppendorf tubes.
Activity-Based Proteomics: Methods and Protocols by Herman S. Overkleeft, Bogdan I. Florea