What would be the possible reason for this:
Following are our PAGE setup;
Protein Source:Rat Liver
Preparation : Ground in Lysis buffer and centrifuged and collected supernatant. Equalised all the samples with lysis buffer and loaded using 5x sample loading dye and beta mercaptoethanol.
Concentration : 30 microgram
SDS PAGE : 10%gel. Run 1.5 hr (120 V)
Sample preparation
Sample buffer (SDS reducing buffer)
3.55 ml deionized water
1.25 ml 0.5 M Tris-HCl, pH 6.8 2.5 ml glycerol
2.0 ml 10% (w/v) SDS
0.2 ml 0.5% (w/v) Bromophenol Blue
9.5 ml total volume
Store at room temperature.
Use: Add 50 µl ß-mercaptoethanol to 950 µl sample buffer prior to use. Dilute the sample at least 1:2 with sample buffer and heat at 95°C for 4 min.
The cassette with both gels is not properly closed. Running buffer is leaking, that is why the level of buffer is at the short plate height. Once you assemble the two gels in the cassette and fill the inside with buffer (to the top), the level of running buffer shouldn't decrease much during the process. In your case, once the running buffer is not covering properly the short plate, the migration is not equal along the gel.
hi
you can creak again running buffer and gels buffer composition and concentration
Hi,
Several factors can cause this kind of error,
1. Check the running buffer pH
2. You can freshly prepare a sample dye and try it once
3. Increase the sample heating time to 6 or 7mins
4. Check the stacking and resolving gel chemicals and its preparation
- Badges
- Science method