The expected fusion protein is 70kda, but it is only 30kda. I used the pet-32a plasmid, and the tag of this plasmid is 17kda. What is the reason? There are not many rare codons, only one or two. Could this be affected by the secondary structure?
Did you sequence your plasmid after ligating the insert into the vector? It could be that you have a truncated or otherwise incorrect insert, such as a frameshift causing a premature stop. The other possibility is that you are getting erroneous initiation from a methionine further on in the insert sequence, so check that your insert is in frame with the initiating methionine.
The other possibility is that your protein does not migrate on SDS-PAGE as per the MW standards, which is common for certain proteins (e.g membrane proteins tend to run slower because of their high hydrophobicity when compared to water soluble MW standards). I am presuming that you purified this protein? You therefore need to confirm that the band in question is your protein, by using western blot or mass-spec protein sequencing etc.
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