Biomedical Engineering Reference
In-Depth Information
decreased dramatically; therefore, potassium should be changed to
sodium by dialysis or gel filtration of the sample.
Mix liquid samples with sample buffer Soln. G and G
in a
1:1 ratio or 3:1 with Soln G
. Then heat the samples to 95
◦
C for
5 min and centrifuge to collect condensed water. If the samples are
stored frozen, heat them again prior to application since the formed
SDS/protein complex is sometimes unstable.
Thesamplemusthaveabluecolor.Ifnot,adjustpHwithsome
microlitersofSoln.B.
If unknown samples have to be analyzed by Western blotting,
it is recommended to run the sample as well as reduced and nonre-
duced, and heated and unheated, since sometimes epitopes are
destroyed when disulfide bridges are cleaved or reconstitution fails
after withdrawing of SDS.
To avoid reduction of an unreduced sample by its neighbor
during electrophoresis, inactivate the DTE overload of the reduced
sample by iodoacetamide or NEM.
The applied sample volume depends on the slot size and should
be as low as possible, and the concentration should be as high as
possible. As an example, with 1 mm gel thickness and 10 to 15 cm
separation gel length, the amount of protein should not exceed
20 - 30
µ
/
mm
2
slot bottom area.
The minimal amount of applicable protein depends on the de-
tection method. Mostly detection by Coomassie staining is tenfold
less sensitive than by silver staining, which drops down to 1 ng per
band.
Add the samples with a syringe (e.g., Hamilton syringe) or
special pipette tips to form a lower layer below the electrophoresis
buffer within the slots.
Start the electrophoresis immediately when all samples, marker
protein mixtures, or references are applied, because molecules dif-
fuse through the soft stacking gel and the pH jump between stack-
ing gel and separation gel, which is important for separation power,
drops down.
Connect the electrophoresis apparatus to the power supply and
g
Electrophoresis
switch on the voltage (time course) directly after sample applica-
tion.
Check the proper connection by wires: In SDS electrophoresis
the migration is “−” (mostly black wire)
“+” (mostly red wire),
when cetyltrimethylammonium bromide (CTAB) is used instead of
SDS the direction is “+”
→
“−”.
As a general rule, a voltage of 30 - 40 volts is useful for intro-
→
Voltage during
electrophoresis
ducing the sample into the stacking gel and field strength of 10 -
15 V
/
cm separation gel length for the separation is sufficient. If an
efficient cooled device is used, the field strength may be increased.
Caution!
Direct current used in electrophoresis is very dangerous.
Never disconnect electrodes from the electrophoresis chambers be-
fore switching off the power supply!
