Hello,
maybe anyone has an idea why this bands looks like below?
This is native page for basic proteins (acidic gel) with reversed electrodes
8 x separating gel buffer: 2,2 M CH3COOH pH 4,0
Separating gel – 8 %
8 x stacking gel buffer: 0,07 M CH3COOH pH 5,0
Stacking gel – 4 %
I prepared gels according to data in the table (with addition of 100 µl 10% APS and 20 µl TEMED - both)
10 x Running buffer: 0,4 M β-Ala pH 4,0
Running conditions: 5 mA until the tracking dye has reached the separating gel, then 25 mA.
Sample: 0,9 mg per well (protein)
I would be thankful for the answer.
Sometimes certain proteins or the presence of salt in the gel sample can cause this effect but the type of gel system you are using is generally more challenging than other methods (SDS-PAGE, IEF, etc.) so this might just be the way it is.
The underlying cause is localized variation in the electric field. The field, E, is determined by the current, i, conductance, k, and cross-sectional area, A as:
E = i/kA
It is variations in k that cause changes in the electrophoretic mobility, u, of the proteins since:
u = v/f' = q/E, where v is the velocity and f' is the effective retarding force (friction, entanglement, etc.), q is the charge on the protein and E is the field.
Localized variations in E can be caused by ionic material diffusing out of the protein band (e.g. salts, protein, thus changing the conductance), and variations in the gel composition (effectively altering A).
I think the voltage is the problem, it happened to me before, and when I started to decrease the V at the beginning, I got better results. You can try to start with 60-75volt for the first 15-20min then you can increase the V.
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