Dear Talha Shafi

for western blotting, since it is a denaturing methods, the position of the his tag is not essential,

for protein purification in non-denaturing conditions generally the Tag is located close to the N- or C-term to improve the probability that it is exposed and accessible to the binding and not buried between 2 globular domains, but it depends from the other domains that are inserted in the construct.

good luck

Manuele

As already said the position in the sequence is not essential for detection in western blot.

However if you want to know which teminus is better for purification, you can use alphaFold2 prediction to check wich terminus is exposed and tag accordingly.

Hi there,

The Histag can be introduced within the protein sequence provided it remains accessible for purification purpose (ie. located within a loop exposed to solvant)

For Western blotting, it mainly depends on which specific antibody you are using: This specific antibody (3D5, https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0220.pdf) requires the His tag to be at the very C-terminus, others are best for N-terminal RGSHis5, other antibodies recognize poly-His sequences independent on whether they are at the N-terminus, in an acessible internal loop or at the C-terminus. Check with the provider of your antibodies whether or not the specific product you are using is OK for your application. For IMAC purification, the main factor is whether or not a sufficient number of Histidine side chains are accessible on the surface of the molecule.

One more point to consider - what protein extraction buffer are you using? More specifically, which detergent is used in the buffer?

With membrane proteins, a detergent such as Triton X-100 will not be sufficient to extract the protein from the bilayer and may result in most of it getting stuck in the insoluble fraction. A more suitable alternative would be something like Triton X-114.