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You shouldn't need the ATG at the start of the GST, but it shouldn't matter.
GST is relatively large, if you are not going to be purifying the protein, then FLAG or V5 are good alternatives. However, GST is very soluble and can stop proteins entering the insoluble fraction.
I don't know about the RE site thing, never heard that before.
The kozak sequence can help, but is not absolutely necessary. The C after the ATG may lower the expression, but this is not necessarily the case.
If you can't see the band - what conditions have you tried for lysis? Do you have a GST +ve control lysate to ensure that the antibody is working? How much protein are you loading? What promoter is on the plasmid?
#149525 C-terminal GST fusion problem in mammalian cell
Posted
on 04 February 2013 - 12:19 AM
