I have for several months tried to purify recombinant flag-tagged proteins expressed in HEK 293 cells using the anti-flag m2 magnetic beads but to no avail. All eight proteins are adequately expressed in cell lysates and are seen at the right sizes by western blot. However, whenever I purify the proteins i do not see the eluted proteins by western blot. I have done several optimisation such as reducing the volume of cell lysate and increasing the volume of packed beads in order to increase the binding capacity but to no avail. The proteins will just not show in the elution fractions but are expressed in the cell lysates. please can I get any suggestions?
First check whether it is binding problem or the elution one? If your FLAG tag is somehow is not exposed to the surface then it will not bind. But I suspect that this is the elution problem. When I elute the protein from the FLAG beads then I keep it for 30 minutes. Did you load the bound beads on the SDS PAGE? Also check your buffers whether they are comparable with the FLAG tag beads.
Hi Ram thanks very much. I use acid elution with 0.1M glycine HCL PH 3.0. I will try loading the bound beads on SDS. Thanks a lot.
As suggested by Ram, please load the IP beads also on SDS-PAGE to make sure the problem is not with the elution.
As suggested by Ram, if the tag is not exposed then it will not bind and get purified. As a backup, try changing the position of the flag tag. for example, if it is at n-terminus then place it at c-terminus or vice versa. hope it helps
If you think the elution step is the problem, you can try using FLAG peptide as an alternative method to get the protein off the antibody. When you blot the IP beads, if you have a problem with the IP antibody obscuring your signal, I suggest using HRP-FLAG antibody as a detection reagent. You can also try rabbit anti-FLAG, though it's never given a very strong signal when I've used it.
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