Electrophoresis Using Excelgel Sds And Buffer Strips - Pharmacia Biotech Multiphor II User Manual

5. Operation
5.1 Electro-
phoresis
using
ExcelGel SDS
and
buffer strips
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This section describes the running procedure for SDS PAGE using buffer
strips. The running of ExcelGel SDS, gradient 8-18 using ExcelGel SDS
buffer strips is chosen as an example, but the basic method is applicable to
all SDS PAGE and native PAGE gels.
ExcelGel SDS, gradient 8-18, is a 0.5 mm-thin, precast polyacrylamide gel
for horizontal electrophoresis of SDS denatured proteins. To facilitate
handling, the gel is cast on a plastic support. During the run, the precast
SDS buffer strips supply the gel with buffer ions. For further information,
see the information supplied with ExcelGel SDS gels.
Sample preparation
Dissolve the samples in sample buffer B (for recipes, see Section 4.9 Stock
solutions). Then heat the sample solution at 95 °C for 3 minutes. The
sensitivity of your development technique and the volume of sample
applied to the gel will determine the lower limit of your sample
concentration. Generally, the sample must contain 200 to 500 ng of each
component for Coomassie staining, and at least 10-25 ng of each
component for silver staining. For molecular weight determination, we
recommend the use of molecular weight calibration kits LMW and
HMW/SDS.
Sample application
In horizontal electrophoresis there are three methods of applying the
sample: application strips, paper pieces and sample wells. Sample
application strips are put on the gel surface, forming sample slots. Silicone
rubber sample application strips are specially designed for easy sample
application.
The following application strips are available:
SDS application strips for up to 40 µl of sample in 26 slots.
IEF/SDS application strips for up to 20 µl of sample in 52 slots.