I'm using a standard SDS PAGE protocol, but since a month or so, some mysterious issues started to cause us trouble as seen in the first two pics (gel1 and gel2). The third picture is from a somewhat better experiment (gel3). From our side, all experiments were prepared under the same conditions.
The first lane in the Gel1 is a protein ladder. The same as in the first lane from Gel3.
Could anyone help us find a common cause for the two bad experiments in the first two pictures attached?
Thanks in advance for any suggestion!
Hi Horea Szedlacsek
the first thing to do is: try to check the compatibility between your running buffer and your gel (pH and compositions). secondly: make sure to check if your gel heated up during electrophoresis.
are these image of standard SDS-PAGE or stain-free?
Things to look on:
- Blurred bands of low molecular weight proteins may be due to the problem on currents/ voltage. The run is too fast because the current is too high, try to decrease the voltage.
- Prepare fresh APS and TEMED, buffer.. etc. and prepare fresh gels.
- After electrophoresis run, immediately go for fixation and staining. Don't leave it in buffer.
- Increase gel % 14-16%.
SDS-PAGE gels rely on a sophisticated discontinuous buffer system, with zones of different pHs and conductivity, which allows stacking of the proteins in a narrow zone before they enter the separating gel. Most problems arise from deviations in the composition of buffers. There are various protocols for preparing and running such gels. I have always stuck to the original protocol of U. Lämmli, Nature 227,680-685 (1970). Beside problems with buffer composition, poor results may also arise if acrylamide is badly contaminated with acrylic acid, which could cause electro-endoosmosis. To avoid this, you could eliminate acrylic acid by storing the acrylamide/ bisacrylamide over beads of ion exchange resin
Thank you very much for your help!
Rabab G. El Mergawy These are coomassie stained gels.
Bhairavi Vajaria We do the fixation and staining immediately after the run. What should I learn if I increase the acrylamide content to 14-16%?
Our buffers are as follows:
Tank buffer 5X:
72g glycine; 15,2 g Tris base; 5 g SDS
Running buffer:
1,5 M Tris pH=8,8
Stacking buffer:
1 M Tris pH=6,8
10% Gels are prepared from a solution containing:
1,9 ml water; 1,7 ml 30% acrylamide mix; 1,3 ml Tris (pH=8,8); 50 ul 10% SDS; 50 ul 10% APS; 2 ul TEMED
Stacking:
680 ul water; 170 ul 30% acrylamide mix; 130 ul 1,5M Tris (pH=6,8); 10 ul 10% SDS; 10 ul 10% APS; 1 ul TEMED
Except for the tank buffer, frames and power source, we also changed completely the ingredients. With mixed outcomes. One typical case is depicted in the attached files. We ran simultaneously a gel prepared with our ingredients (Gel4) and ingredients from other groups (Gel5) at 60 mA. The sample are identical in both cases and the first lane is LMW ladder. One might find it strange that a pair of two 10% 1,5 mm gel runs at 60 mA for ~ 2 h until the front comes out? Any ideas what the problem might be?
From the photos you show, seem to me the environment within which you conducted the electrophoresis caused all these. First, maybe about your running buffer. Although it's typically okay to re-use that, but whenever the pH changes it creates problem, so better 3 time use is maximum. Second, maybe the voltage, you can try to lower that a little bit. Hope this helps.
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