Dynabeads His Tag

Go to the profile of j1234t1234
May 12, 2017
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I performed a co-IP with a His tag protein and a flag tag protein using the dynabeads His-Tag. From doing co-Ip experiments using both flag and HA antibodies I have established these two proteins associate. When I performed this experiment using the dynabeads His-Tag ( I wanted to use this method because I want to use the alliance for a native gel) I found when through immunoblot analysis probing with the Flag antibody the negative control sample which is just transfected with the Flag construct, had identical levels of Flag as the samples that were co-transfected with both the HIS and Flag tag constructs, thus the beads are binding to nonspecific proteins. Can anyone give me some feedback to alleviate this problem.

2 Comments

Go to the profile of mdfenko
mdfenko 8 months ago

are you able to try to bind the untagged protein to the dynabeads? you may find that there are exposed his residues on the native protein.

you may have to adjust the ionic conditions to reduce non-specific binding.

Go to the profile of relaxin
relaxin 8 months ago

Your Flag-tagged protein may contain histidine-rich clusters in its sequence, so it may bind to Ni-resin nonspecifically. Do you have its protein sequence to check this?
Wash the protein-bound Dynabeads with a buffer containing 25 mM imidazole may reduce the nonspecific binding.