I am purifying a heterotetramer, with one subunit having a 1D4 tag and the other having a FLAG tag. I purified from Sf9 with HBS pH7.3 and 0.002%LMNG first with Anti-1D4 resin and got an adequate amount of protein (checked with SDS PAGE and FSEC). I incubated my 1D4 elutions with Anti-FLAG beads and tried to elute with 0.1mg/mL 3xFLAG peptide, but there was no protein in this elution. I checked the concentration of my flow through fraction as well, and there is some protein but not all that was in the 1D4 elution. I was wondering if anyone has any insights into how this could be.
There are several possibilities.
1. Your complex did not bind to the flag beads and was removed with the washes. Run a sample from the solution after incubation with beads to check that. Use a positive control to make sure your beads are ok.
2. Your elution is not working. Use a positive control to make sure your flag peptide is OK
3. Your binding conditions caused the complex to sediment. If you are using the beads as a batch, it will be in the beads fraction. Boil the beads in sample buffer after elution and run to see whether your protein is still there. If you are using a column, sedimented protein can be stuck in the column or washed in the flow through. Run a sample. For diagnosing small samples you can do a Western with anti flag antibodies.
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