Overview of minipurification

Minipurification rapidly separates viruses from host proteins allowing 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. Samples must be sufficient to give stable pellets. After dissolving pellets 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. Heating extracts eliminates contaminants, but degrades virus only above 50C. Thiols activate host proteases and should be avoided.

Normally gel electrophoresis requires ~1% of the sample. The remainder can serve for further diagnoses (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.

The minipurification protocol

Factors influencing minipurification

When is minipurification useful?

Potential questions about minipurification?

Examples: Maize viruses - MDMV, WSMV, MCMV: Wheat viruses - WSSMV?, SBWMV
Sugarbeet viruses - BSBMV: Soybean viruses - BPMV, BCMV
New! - Improving minipurification