A protocol of coupling Ab to the magnetic Epoxy Dynal beads M270
This protocol is adapted from the original protool from Invitrogen and from the protocol used in Brian Chait’s lab at the Rockefeller University. The protocol is optimized for coupling 50 mg of epoxy Dynal beads to 500 ug of Ab. This amount of beads is usually enough for 5-10 IP experiments.
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A protocol for purification of proteins tagged with a 3xFLAG-6xH double affinity tag
1. Grow X liters of cells overnight to the density 2-5×10e7 cells/ml
2. Spin down cells in 1 liter bottles at 5000rpm 15-20’ 4ºC
3. Remove supernatant and collect cells with up to 50ml of ice cold water into 50ml conical tube.
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A protocol of IP from mammalian cell cultures
1.Culture 20 flasks (Corning, 430641; 75 cm2 culture area/flask) of mammalian cells until reach over 95% confluent.
2.Remove the culture medium, and rinse the cells twice with cold 1X PBS and aspirate.
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Cryogenic disruption of yeast cells
This is a protocol for cryogenic disruption of cells. Our protocol is a modification of the protocol designed by Markus Kalkum. Also, here you can find the the original paper first describing the use of cryogenic disruption of bacteria cells.
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A protocol for using Modular MS tool
This protocol described the major steps in using two mass spectrometers prOTOF and vMALDI-LTQ for measurement of MS, MS/MS and MSn spectra. The analysis starts with obtaining highly accurate single-stage MALDI spectra in the prOTOF mass spectrometer. Then, the MS/MS and MSn spectra of all detected species can be obtained in the vMALDI-LTQ mass spectrometer.
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In gel digestion protocol
This protocol describes a procedure for identification of proteins separated by SDS-PAGE. This protocol is based on several “classical” protocols and optimized for the efficient extraction and characterization of the tryptic peptides.
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On beads digestion protocol
This protocol describes a procedure for identification of proteins purified using 3xFLAG-6xH or 3xMyc-8xH double affinity tags. Protein and protein complexes can be directly digested on the metal chelating resin. The proteolytic peptides can be analyzed in our modular MS tool or in an HPLC-MS&MS/MS tandem mass spectrometer.
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I-DIRT, a general method for distinguishing between specific and nonspecific protein interactions
This protocol describes the I-DIRT technique that helps to distinguish contaminants from the real interactors in immunopurified complexes. The technique was designed by Alan Tackett when he worked at Brian Chait’s Lab at the Rockefeller University.
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