One of my cloning results indicated a nucleotide exchange from G to T and the resulting aminoacid exhange is Arginine instead of serine. How can I find what effect can it possibly have at the protein level?
The only way you can make a meaningful prediction is to take a look at the structure of your protein (it does not have to be a model from high resolution X-ray diffraction data, a good homology-based model will do) and then examine what interactions the original residue was involved in. If you do not have the expertise, get hold of the friendliest structural biologist in the neighborhood and have her/him do it for you.
Dear Nilima,
it is difficult to predict a priori the effect of an amino acid substitution in a protein. Firstly you can use bioinformatic prediction tools, such as panther, SIFT, SNP and GO, polyphen-2. But the in silico prediction can give you just some suggestions.
To determine if your protein is affected you should perform, if possible, a quantification and a functional assay. The quantification through Western blot or ELISA or similar is important since the substituion can affect protein stability and thus protein levels. The functional assay gives you a measurement of the mutant protein activity, but this can be performed for a limited number of proteins (enzymes, transporters). Also localization can be affected, as well as the binding with other subunits in case your protein makes part of a complex.
Try to blast it, it might be a naturally happened isoform. It is unlikely to introduce G to T mutant by PCR, if it is not in the priming region. PCR generally introduce G-A, or C-T mutant. Spending time to sort out the unsure possibility, it would be easier just changing it back to what you expect.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins