GE PhastSystem User Manual page 90

Trouble Shooting Guide
Symptoms
Loss of protein
bands/
appearance of
extra bands
Streaking
Proteins only runing
half the gel
Background smear
on the gel
6
Probably Cause
1. Proteolysis of sample proteins.
2. Incomplete protein dissociation
with SDS-PAGE.
1. Poorly soluble proteins.
2. Particulates in the sample.
3. Impure SDS in the sample.
4. Samples stored frozen with SDS
and not warmed prior to electro-
phoresis.
5. Sample overloading.
6. Dirty sample applicator.
1. Frozen buffer strips.
2. Not good enough contact be-
tween buffer strips and elec-
trodes.
1. Touching buffer strips with
fingers.
Solution(s)
1. Prepare samples at low tempera-
ture. Try adding protease inhibi-
tors.
2. Heat samples in SDS-buffer (10
mM Tris/HCl, 1.0 mM EDTA,
pH 8.0 and 2.5% SDS, 5% ß-
mercaptoethanol) at 100°C for at
least 5 min.
1. Apply the sample under low cur-
rent e.g. 1.0 mA. Increase the
separation time.
2. Centrifuge or filter the sample.
3. Use analytical grade SDS (99%
pure) to denature sample pro-
teins.
4. SDS precipitates upon freezing;
warm samples to 20°C before
loading sample applicators.
5. Dilute the sample.
6. We recommend that sample ap-
plicators be used only once.
1. Do not freeze at any time! Frozen
strips lead to proteins only run-
ning half the gel, more blurry and
a very low ending current.
2. Always check the contact and
gently bend the electrodes down a
little if necessary.
1. Avoid touching the buffer strips
with anything when using sensi-
tive staining methods.
Wear gloves.