THIS IS HOLDING TEXT Classic discovery proteomics is most commonly carried out using data-dependent acquisition (DDA) strategies. While it is easy to setup data acquisition and analysis to obtain a high proteome depth, the reproducibility is commonly low due to stochastic sampling. In contrast, data-independent acquisition (DIA) allows for more accurate and sensitive protein quantification and has become widely used. Our iST kits are suitable for experiments with metabolic labeling or in label-free mode, whereas our iST-NHS kits are suitable for experiments with chemical labeling such as iTRAQ or TMT.
Add 50 µL of our LYSE or LYSE-NHS buffer directly to the cell pellet and proceed with regular protocols
If the volume of FACS sorted cells exceeds 11 µL, centrifuge to take off the supernatant and decrease the volume before adding lysis buffer. Alternatively use the higher concentration lysis buffers.
Certain lysis buffers are compatible with our iST and iST-NHS workflows. For further details please see our FAQ 1.1 If they are not compatible protein precipitation may be required.
If extracellular matrix or transmembrane proteins are not of interest, you can use cell culture-grade trypsin to detach the adherent cells, wash them once with PBS and then store the cell pellet fraction as input for our regular protocols. Alternatively, if extracellular matrix or transmembrane are of interest and affected by partial trypsin digestion, add 50-100 µL of our LYSE / LYSE-NHS buffer directly to the cells and scrape them off using a rubber policeman. Collect the scraped material and heat to 95°C for 10 min before continuing with the regular iST / iST-NHS protocol.