Web-based pathway analysis tools
- Reactome– Pathway database
- EnrichR– Analyses for Pathways, Ontologies, Diseases, Cell types
- GOrilla– Enriched GO terms
- KEGG– Pathway analysis
- DAVID– Pathway analysis
- Panther– Gene Ontology analysis
- STRING– Interactome analysis
- WikiPathways– Pathway database
- KSEA– Tool for kinase activity inference from quantitative phosphoproteomics
- Human Protein Atlas– Human protein expression map integrating data from multiple sources
- Proteomics DB– Human tissue-dependent protein expression map
Volumes of lysis buffer must be determined in relation to the amount of starting material. As a rule of thumb 5 to 10 volumes of your centrifuged pellet should be sufficient. Protein extract should not be too diluted to avoid loss of protein and large volumes. On contrary, too little lysis buffer will impair the lysis efficiency. The optimal concentration of total protein in the lysate is 1–5 mg/mL.
If you are unsure about the lysis buffer volume, perform a pilot experiment by lysing the sample in a small volume. Based on the results you can dilute the sample or repeat with a larger volume.
As a starting point it is recommended to use approx. 500.000 cells (varies depending on the cell type)
Example of cell lysate preparation
- 2% SDS in 100mM Tris pH 8 supplemented with protease inhibitors
- Add lysis buffer and pipet the cell pellet up and down vigorously
- Place the sample in a heating block (90oC, 1000rpm, 10min)
- Spin down droplets
- Sonicate the sample to shear the DNA (5 min, cycle 30/30 sec). ALTERNATIVE: Completely degrade all nucleic acids by Benzonase. Check the viscosity of the sample to ensure that the chromatin has been completely degraded. If viscosity persists do not proceed and contact us.
- Centrifuge for 10 min at 20.000 × g, remove cell debris if visible. Measure protein concentration by your assay of choice (check for buffer compatibility)
- Prepare aliquots of 20ug total protein and snap freeze; store in -20oC until further use.
- Clearly label the tubes and bring 2 aliquots (frozen) to the faciltity (one to be analysed and one as back-up)
For gel-based approaches
- Always wear gloves
- Leave blank lanes in between samples to avoid any spill over effect
- Use MS-compatible gel staining (no fixation or microwaving)
- Wash the gels extensively after the end of the run to remove as much remaining SDS as possible.
- Store the gels in clean boxes (pre-rinsed with organic containing solution) to minimize keratin contaminations.