GE PhastSystem User Manual page 70

6. Evaluation and presentation of data
70
Table 2: Molecular weight calibration kits available for native and SDS electrophoresis.
Marker MW range
kit
HMW kit SDS: 18,500-
330,000
Native 67,000-
670,000
HMW SDS SDS 53,000-
kit 1 212,000
LMW kit 1 SDS 14,400-
94,000
PMW kit 1 SDS 2,512-
16,949
1
Designed for use with SDS electrophoresis only
Procedures for MW measurement
Each MW calibration kit is supplied with complete instructions for use.
Follow these instructions, but dilute the vials with 200 µl of buffer for
Coomassie staining, and with 3 ml of buffer for silver staining, instead
of 100 µl as suggested in the instructions. A condensed version of the
instructions is given below to illustrate the simplicity of the method.
1. Dissolve the calibration kit proteins in 200 µl (for Coomassie
staining), or 3 ml (for silver staining) of suitable buffer for elec-
trophoresis.
For native-PAGE: reconstitute one HMW calibration kit vial in distilled
water.
For SDS-PAGE: reconstitute one HMW or LMW (or both) vial in
10 mM Tris/HCl, pH 8-0; 1.0 mM EDTA, with 2-5% SDS and 5-0%
ß-mercaptoethanol. Mix by gently swirling. Heat this mixture at 100°C
for 5-10 minutes.
Reconstituted, denatured kit proteins can be stored frozen at 20°C.
1. Prepare the sample proteins in the appropriate buffer as given
above.
3. Carry out electrophoresis according to the method given for
native or SDS-PAGE in the Separation Technique File.
4. Develop the protein bands using one of the techniques in the
Development Technique File.
5. To easily measure the band distance, mount the gel in a slide
frame and project the image to the desired format using a slide
projector. Alternatively, scan the gel with PhastImage.
6. Measure the migration distance of the calibration proteins and
calculate their R
R , = distance of the band from the origin
f
distance from the origin to the reference point
µg marker/
number
Number of prot. per
vials kit vial
10 250/5
10 200/5
10 600/6
1 2000/5
, values:
f
Storage
Unused Reconst.
+4°C 20°C
-20°C 20°C
+4°C 20°C
+4°C 20°C