Bio-Rad Whole Gel Eluter and Mini Whole Gel Eluter - Manual (Page 23)

7.5 Prepare Continuous Gels
(10 ml acrylamide monomer solution)
% T Deionized Gel buffer Acrylamide/Bis solution
(acrylamide H
2
O solution* 30% stock (37.5:1)
monomer) (ml) (ml) (ml)
4% 6.65 2.00 1.33
5% 6.30 2.00 1.67
6% 6.05 2.00 2.00
Catalysts 10% APS* TEMED*
Resolving Gel 50 µl 5 µl
Stacking Gel 50 µl 10 µl
* Amounts are per 10 ml gel volume. To make 10% APS, dissolve 100 mg in 1 ml of deionized water. TEMED is used neat.
Below pH 6, TEMED becomes less effective as a catalyst. Between pH 4 and pH 6, increasing the concentration of TEMED
5-fold to 10-fold will polymerize the gel.
7.6 Sample Preparation
Sample buffer for continuous native PAGE is a dilution of the electrophoresis buffer.
The concentration of the sample buffer is generally 1/10 that of the running buffer. Glycerol
is added to the sample buffer to a final concentration of 20%.
Section 8
Troubleshooting Guide
Problem Cause Solution
1. Incomplete elution of a. Air bubble formation a. Refer to Section 3.2.
proteins from the gel. between the anode
and cathode.
b. Buffer or running b. Refer to Section 4.
conditions are not
optimized.
2. No detectable proteins a. Insufficient protein a. Increase total protein
in collected fractions load. loaded. Use silver stained
(complete elution). SDS-PAGE gels to
analyze individual
fractions.
b. Protein adhering to the b. Reverse current for
cellophane membrane. 10 seconds after the
elution run. Decrease the
run time.
3. Cross-contamination a. Elution chamber core a. Tighten the hex screws
of fraction. not tightened down. in a crisscross pattern.
b. Filter paper or cello- b. Refer to Section 3.2
phane membrane (assembly).
missing or placed in
wrong order.
21