Overview of minipurification
Minipurification is a small scale "universal" plant virus purification protocol.
Minipurification depletes host proteins and concentrates virus from extracts of infected plants, facilitating detection of capsid proteins by SDS gel electrophoresis. Citrate buffers extract most viruses. Brief ultracentrifugation "clarifies". Ultracentrifugation of supernatants pellets virus. Pellets contain some rubisco and other host proteins as well as host polysaccharides. Samples must be large enough to give stable pellets. Pellets are dissolved in dilute phosphate buffer; aliquots are diluted into "cracking buffer", heated, and electrophoresed on SDS gels which are then developed with silver.
Nonhost protein bands indicate virus infection. Band size and intensity facilitate identification. The method is quicker than dsRNA purification and works for a wider range of viruses.
Protein patterns are insensitive to extraction pH between 5.5 and 7. Higher citrate concentrations give cleaner preps. Note that higher citrate concentrations not only minimize electrostatic interactions, but stabilize virues. The stabilization reflects the position of citrate in the Hofmeister series. Heating extracts eliminates some contaminants, but degrades virus only above 50C. Avoid thiols because they activate host proteases.
Normally gel electrophoresis requires ~1% of the sample. The remainder can be analyzed further (EM, serology, inoculation to plants, etc.).
Variants of the procedure (e.g. heat, proteolysis, changing pH, salt concentration, buffer, etc.) are useful in developing purification schemes and in identifying specific viruses.