I'm having trouble with my Western blot results, and I'm hoping to get some advice. I used a 4% stacking gel and an 8% resolving gel to detect GAPDH (36 kDa). The SDS-PAGE was run at 120V for 2 hours and 30 minutes. However, the bands came out smudged and unclear, as shown in the attached image. What could be the reason for this issue? Could it be related to the gel concentration, running conditions, or another factor?
8% is a very soft gel. It's difficult to handle and prone to deforming when pressure is applied, as in Western blotting. If you can use a higher percentage gel, and still have the bands you care about be within the gel, the result may be somewhat better.
To avoid the gel becoming distorted when placed on the membrane, you can float it on. Submerge the membrane in transfer buffer, them float the gel into place onto the membrane.
there are a number of reasons causing this result: 1) using 120 voltage and 2 and half hours for 8% gel is to long and would generate heat to damage your proteins; 2) when your made the gel, you didn't mix well, so gel solidification and SDS concentration in the gel were not even; 3) because the concentration of this gel was so low and soft, when you transferred proteins from gel onto membrane, the sandwich could make the gel to change its shape.
- Badges
- Science topic
- Similar topics
- Journal Impact Factor