We use double affinity tags, 3xFLAG-6xH or 3xMyc-8xH, for purification of proteins and protein complexes (see our IP protocol). The use of 3xFLAG or 3xMyc tags are usually sufficient to obtain highly enriched protein complexes. However, an additional tandem purification step can be very useful to remove an excess of the elution peptide and concentrate the complexes on the metal chelating resin. After the final wash of the chelating resin, the purified proteins can be efficiently eluted for separating by SDS-PAGE or left on the beads for on bead digestion.
Here is the protocol for digestion of proteins and protein complexes on the metal chelating resin.
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1.Wash the cobalt (or some other metal) chalating beads two times with 1 ml of 50mM ammonium bicarbonate buffer. The purpose of this step is to remove the detergent used in the IP buffer as completely as possible.
2. Add 5-10 ul of 1pmol/ul trypsin solution in 50 mM ammonium bicarbonate to the beads. Incubate 5-30min; collect the supernatant and incubate it further for 5-12 hours. Limited digestion may help to minimize elution of nonspecifically bound proteins (this still remains to be proven). Otherwise, add trypsin and digest on the beads all the time (6-12 hours)
3. Analyze 1/3 or ½ of the digest (3-5 ul) directly by a MALDI modular MS tool or HPLC-ESI.
Note: Filter or spin down the digest before loading a portion into an HPLC column. This step is necessary to minimize the chances of blocking the HPLC tubings with small micropaticles of metal chelating resin.
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