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I have been meaning to add some links for Protocols, Methods and SOPS. One useful web resource I encountered is to the BioTechniques Molecular Biology Techniques forum. There is just a ton of information, protocols and the like. I haven’t had a chance to play around with it too much, but this looks like a good resource to book mark.
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Reasons for or against putting a hist tag at both ends?Post new topic Reply to topic BioTechniques® Molecular Biology Techniques Forums Index -> Protein
PostPosted: Nov 16 2007 7:37 am Post subject: Reasons for or against putting a hist tag at both ends? Reply with quote
I ordered a custom made NusA fusion vector from Addgene recently and originally thought that the vector had only a C-terminal histidine tag. Upon closer inspection I noticed that there is also a N-terminal histidige tag. The C-tag is naturally optional so that by including a stop codon in your insert you can choose not to have it. My question is that is there especially something for or against using both tags? We can assume that the tag sequence does not affect the specifity of my assay and doesn’t need to be removed. The tag(s) are there for purification.Question 2: Do you know if there is an actual difference in leakage between using TAA versus TGA as the stop codon in bacterial expression?
PostPosted: Nov 16 2007 10:42 am Post subject: Reply with quote
There is no reason to use both tags. I think the vector is designed that way, so someone can have a choice of putting the tag at the N-terminus or the C-terminus, depending on the protein being expressed. There may be restriction enzyme site upstream of the N-terminus His-tag to let you delete the tag sequence, if you so chose.It is better to avoid TGA stop codon when you express protein in E. coli to avoid read through. Here is an example.
http://www.biochemj.org/bj/309/0411/3090411.pdf
Just like human, they go through STOP sign. Laughing
