Protein resolution

TABLE 4.2 Comparison of Protein Resolution Factors (Rs) on TSK-GEL SW Columns"... 

Two sets of soluble globular proteins were selected from Protein Data Bank (PDB) for testing for false positive results. In the set SOLUl of 187 such proteins resolution for each protein was equal or better than 3 A. Secondary structure conformations were determined by the DSSP algorithm. In the set SOLU2 of 147 proteins (protein data set used in [7] plus 21 additional proteins) only proteins known with equal or better than 2.5 A resolution were included. There was less than 25% pairwise similarity. Three different secondary structures were determined as described by Rost and Sander [7]. Both data sets are available in the Supplementary Material. 

Figure IB demonstrates the Increased protein resolution obtained by adding 6M guanidine hydrochloride In a sodium phosphate elution buffer to separate a standard mixture of proteins. Under the denaturing conditions described, all of the proteins below 67 kDa are completely resolved from each other. Table 3 reveals that the values obtained In the guanidine...

Apparently the protein resolution obtained In SDS Is markedly dependent... 

Several organic solvents have also been employed for SEC of proteins under denaturing conditions. Swergold and Rubin (22) have described a system employing a TSK 3000PW column eluted with 36-45% acetonitrile In 0.1% trlfluoroacetlc acid (TFA). The protein resolution observed In this eluant Is markedly dependent on acetonitrile concentration and requires the low pH provided by TFA. The authors report that under optimum conditions, a linear fractionation range Is observed with proteins and peptides ranging from 300-130,000 Da. 

Many cereal endosperm storage proteins—especially glutelins— are, in their native forms, disulfide-bonded oligomers or polymers. Even if soluble, such proteins may resolve poorly upon RP-HPLC and elute as broad peaks [22], in part due to their many possible polymeric forms. Reduction is necessary to release disulfide-bonded subunits from cereal glutelins or oligomeric prolamins for analysis [19,32,42,76]. Thus, protein resolution, extractability, and stability are often improved by extraction under reducing conditions. To prevent reoxidation and further enhance resolution, resulting cysteine residues may be stabilized by alkylation, usually with 4-vinylpyridine [19,21,29,70,76]. 

Protein Resolution (A) 5uper secondary structural motif Ref. 

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