Hoefer IEF100 - Manual (Page 58)

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General Guidelines
• Sample preparation procedures are being refined and standardized.
It is best to consult the literature to determine if a particular sample
preparation buffer is recommended for the sample type.
• IEF works best with pure protein samples that are solubilized and
denatured, and free of interfering molecules.
• Remove insoluble material with centrifugation.
• Keep the salt content as low as possible.
• Use freshly prepared reagents of high quality, or reagent solutions
that have been stored frozen.
• Do not leave urea solutions out at room temperature for extended
periods of time.
• Never heat protein samples in urea solutions. Heating cause carba-
mylation of proteins and will alter the native charge of the proteins.
• Keep samples on ice to prevent degradation.
• Add protease inhibitors to prevent protease activity. Protease
inhibitors such as PMSF or Pefabloc can be added to inhibit serine
protease activity while pepstatin can inhibit aspartic proteases.
• Some protocols use basic carrier ampholytes or Tris to get a high
pH in the sample buffer. This helps solubilize some proteins and
lowers enzyme activity that would attack the proteins.
• DNA and RNA can frequently be removed by ultracentrifugation.
The sample can also be treated with DNase and RNase solution to
break down the contaminants.
• Keep in mind that some protease inhibitors, DNase and RNase are
proteins themselves and may show up on a 2D map.
• The water used for making reagents should be the highest quality
available. Water with a resistivity of >5 megaohm-cm is best. Water
purified by reverse osmosis or deionized water is acceptable.
Note: Salts are perhaps the most
common contaminant causing poor
IEF results.