Micropreparative high resolution purification of proteins by a combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and membrane blotting.

This research presents an effective, affordable method for protein purification from cells. Processing involves steps of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing, and membrane blotting. The technique’s viability is confirmed by purifying proteins of various sizes from a Sarcocystis neurona lysate.

Methodology

• The process begins by separating proteins from cell lysates using standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotting these onto a protein-blotting membrane.
• The blotted proteins are then stained with Coomassie blue or treated with immunoblotting to make specific proteins visible.
• The specific protein bands identified through immunoblotting are then cut out from the stained blots and put through isoelectric focusing to separate proteins based on their different isoelectric points (pI).
• This focused gel is then stained with Coomassie blue. After staining, the bands are cut out and subjected to another round of SDS-PAGE separation and then electroblotted back onto the protein-blotting membrane.

Results

• After the second round of electroblotting, proteins are sufficiently purified for further study, including microsequencing.
• The efficacy of this new approach was confirmed by purifying proteins with molecular weights ranging from 19 to 100 kDa using a lysate from Sarcocystis neurona, a pathogenic protozoan causing equine protozoal myeloencephalitis.
• The purity of the proteins obtained through this method was shown using reverse-phase high-performance liquid chromatography, which measures the relative quantities of proteins based on their hydrophobicity and polarity.
• Partial sequences of the purified proteins were obtained through N-terminal or digestive sequencing, indicating that the proteins were effectively purified for these types of analysis.

Conclusion

• This research presents a straightforward, cost-effective, and efficient procedure for protein purification from cells, providing a significant contribution to biological and biomedical research methodologies.