1-Which netto charge does a protein have at a pH that is lower than its IEP, and which charge does it have at a pH that is higher than its IEP?
* I already know that at high pH values, the net charge of most proteins is negative but is it different in comparison with IEP?
2- Is the iso-electric point of GFP 5.58 ?
https://chem.libretexts.org/Courses/University_of_Kentucky/UK%3A_CHE_103_-_Chemistry_for_Allied_Health_(Soult)/Chapters/Chapter_13%3A_Amino_Acids_and_Proteins/13.1%3A_Amino_Acids
https://www.sciencedirect.com/topics/engineering/isoelectric-point
If you're at the isoelectric point, the protein is neutral. If you decrease the pH, you increase the H+ in solution, causing side chains to become protonated, so you are either making negative charges neutral (like O- to OH) or neutral charges positive (like NH2 to NH3+), so when you are at a pH below the pI, the protein has a net positive charge. Increase the pH, you decrease free H+, side chains get deprotonated (OH goes back to O-, NH3+ goes back to NH2) so your protein will have a net negative charge at a pH above the pI. You can calculate the pI of any protein using a calculator (https://web.expasy.org/protparam/) but keep in mind this is just the theoretical pI. The actual pK of side chains in a protein is highly dependent on the environment so the calculated pI of a protein will vary from the actual pI. For the most part, you can use the theoretical pI as a good estimate for things like what pH you should use for ion exchange chromatography steps.
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