Evaluation - GE PhastSystem User Manual

6.2 Evaluation

Common
staining
technique
fluorescent detection
Coomassie or
Coomassie-like dyes
green dyes
silver
PAS (glycoprotein)
Paper
A medium to hard paper with glossy finish will give best results for
black and white photographs.
Procedures for measuring the isoelectric points and molecular weights
of proteins, using calibration proteins, are described. A brief discus-
sion about evaluating PhastGel media with PhastImage is presented at
the end.
Isoelectric point measurement
Isoelectric points (pI) of proteins are conveniently and accurately
measured using calibration proteins. Calibration proteins indicate pH
gradient profiles in gels. By measuring the distance of a sample
protein from a reference point to where it focuses, its pI can be
interpolated from the pH gradient profile.
Three pI calibration kits are available from GE Healthcare
(Table 1). Each kit contains 8-11 proteins, depending on the kit.
Table 1: Selecting the correct pI calibration kit for PhastGel IEF media
pI
PhastGel calibration
IEF kit
PhastGel
IEF4-6.5 Low pI
PhastGel IEF
5-8 High pI
PhastGel IEF
3-9 Broad pI
Procedure for pI measurement
Instructions for pI measurement in PhastGel lEF media are given
below:
1. If you know the approximate pI of the protein of interest, select a
suitable PhastGel IEF gel. If it is unknown, use PhastGel IEF 3-9.
2. Use the appropriate separation method for IEF described in
Separation technique file No. 100, chapter 9.
6. Evaluation and presentation of data
Type of
illumination
UV
fluorescent
fluorescent
fluorescent
fluorescent
pI range
covered
by the kit
2.80-5.85
5.85-10.25
3.50-9.30
Type of
filter
yellow or orange
deep yellow or red
(try medium-red with
Panchromatic film)
red
medium-red
blue or orange
Number of
markers for
PhastGel interval
4 (4.15-5.85)
4 (5.85-7.35)
10 (3.50-8.65)
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