О• M,Tyr,Оµ M,Trp,Оµ M,Cys are molar extinction coeffcients of tyrosine, tryptophan and Cysteine residuesДЄ,b,c are number of each type of residues per molecule of a protein.ДЄt 280nm the molar extinction coefficients are: Cys=120, Tyr=1280 and Trp=5690. Molar extinction coefficient of a protein can be calculated from the molar extension coefficient of amino acids Cystine, Tyrosine and Tryptophan using the following equation according to Gill and Hippel,1989 Оµ M,Gdn.Hcl = aОµ M,Tyr+bОµ M,Trp+cОµ M,Cys This is helpful in determining the topology of protein as stated by Nakashima and Nishikawa, 1994 Anders et al, 2001. Total number of Aspartic acid (Asp) and Glutami acid (Glu) are used to calculate total number of negatively charged residues. Total number of Arginine (Arg) and Lysine (Lys) are calculated for number of positively charged residues. Positively and negatively charged residues: There is a significant correlation between pI of a protein and pH of the buffer being used for crystallization, that buffer pH equal to or very near to the pI value offers a reasonable probability of yielding crystals (Kantardjieff and Rupp, 2004). The pI is useful property of a protein to consider for optimized crystallization. The isoelectric point of a protein is an important property because it is at this point that the protein is least soluble.

When the solution pH is below the pI value the protein is positively charged and if it is above the pI value the protein is negatively charged. The computed isoelectric point will be useful for choosing a buffer system for purification and crystallisation of a given protein. Isoelectric point (pI) is the pH at which net charge of the protein is zero. PEPSTATS tool calculates the net charge of a protein assuming the protein is in a solvent having neutral pH (Rice et al. The net charge of a protein is the arithmetic average of charges of all residues present in the protein and depends on solvent pH. For the elution of your protein from the column, you can use either pH gradient or linear ionic strength gradient.The molecular weight is calculated as the sum of atomic masses of all atoms in the molecule. Therefore, you can generate either net positive charge or net negative charge on the protein and select weak cation exchanger (CM-Sephadex) if protein is positively charged or weak anion exchanger (DEAE-Sephadex) if protein is negatively charged for efficient binding of your protein to the ion exchanger. In order to get complete protonation/ deprotonation of these ionizable groups, pH should be 2 units lower or higher than pI of the protein. On the other hand if pH of the buffer is higher than pI, protein will bear negative charge due to deprotonation of these groups. Remember if pH of the buffer is lesser than pI, protein will acquire positive charge due to protonation of the ionizable groups (carboxyl - alpha, beta and gamma or amino - alpha, epsilon, imidazole and guanidino). If you want to use it for purification purpose using ion exchange chromatography, you can nuse the approximate value as well.

Yes, you can do isoelectric focussing using ampholines (gel) of different pH ranges in order to get the correct value of the isoelectric point (pI).

Calculate pi of protein