I have been trying to the run sds-page gel for quite sometime now. But I am facing the same problem repeatedly. while running the dye front runs to the bottom of the gel but after staining I find that both the marker and the samples have run only half the gel. I have been running 15% gel and my protein is of 15kD. I have tried changing all the chemicals but to vain.
check if you r mixing ur resolving gel solution well before pouring into the plates. Does your dye front runs very fast ? what Voltage/Amp you are using ?
I am using 120 V and 25mA current. the dye front takes about an hour to run. Is it okay??
That seems ok.. I assume ur acrylamide solution is not too old or is in good condition...
Dear Kanwaki
Please apply the following comments:
1- Elongate the polymerization times
2- Use 30 V for first thirty min to proteins reach to resolving gel. Then, use 120 V.
3- Allow the front of dye is insert to the buffer.
4- Also, check the value of Acrylamid/Bis acrylamid that used for preparing stacking and running gels.
Cheers
Dear miaomiao,
is it necessary to boil all the samples whether beta mercaptoethanol has been added or not and also the marker..
Dear Masood
I am using acrylamide/bis acrylamide in the ratio of 30:0.8. Acrylamide is of SRL make (extra pure) while Bis is of Himedia make. Are they okay??
The Acrylamide/bis acrylamide ratio can also afect the protein separation. 30:0.8 is an standard relationship, but you can also try 37.5:1.
After you add beta mercaptoethanol you have to boil all your samples and marker.
not any explanations many are there..use good quality acrylamide..APS should be freshly prepared.make sure that you have added 10% SDS.use stain and the sample in 1:1 ration use 1X lamelli stain.dont forget to boil your sample +stain at 94C for 6-7min, initially set the temperature at 50V when the band is crossed the stacking gel change the voltage to 100V.if again the proteins are not separating well then the problem might be with your protocol.you will have to standaridise your protocol
With 15% acrylamide gels it is normal that dye runs much faster than proteins. Reduce acrylamide content to 12%.
Muneeb is right in some way.. your protein marker is not run properly because you run 15 kD protein on 15% gel. Don't do anything you just reduce your resolving gel concentration up to 10 to 12 %. definitely you will get better result.
As suggested by others as well reduce the concentration of acrylamide to 10 or 12% and the markers will move. You cannot expect the marker also very close to the dyefront!
Your detailed description seems for me like something I sow last Millennium, so treat it with the respect to my memory;)
When we were adjusting the pH of the SDS-PAGE Electrophoresis Running Buffer, the result was similar. This is known lab-practice, some pH electrodes got crazy with TRIS.
Here is the recipe for (5x) SDS-PAGE Electrophoresis Running Buffer
(1x: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH8.3)
for the preparation of 2l
30.3 g Trisbase
144.0 g glycine
10.0 g SDS
No need to adjust pH
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