Hello everybody,
I have a problem with SDS-PAGE and I do not know why my samples stop at the top of the resolving gel and they do not run through it. The percentage of gel is 15% and the size of the protein is 25 KDa. They are samples after purification with imidazole and I purified these samples and other similar samples with the same protocol and method of preparing the gel and performing SDS-PAGE successfully (I did not change buffers or other things).
Any advice and suggestions will be greatly appreciated.
I would guess that you have been using a Tris Glycine buffer rather than a Tris Glycine SDS buffer, check whether your SDS in your buffer has precipitated or not, or otherwise try to make a fresh buffer and run your samples again.
Hello Sara
Check the pH of the running buffer. Make sure that your sample preparation is proper. Denaturation of the protein samples after addition of the protein loading dye at 95-100 deg for 5-10 mins is also an important step. Heat ensures that the protein samples are completely denatured.
Does your loading buffer contain reducing agents like beta-mercaptoethanol or DTT in addition to SDS?
I hope this will help.
Regards.
Hi Sara,
make sure you correctly denatured your sample with b-mercapto and also boiled long enough... by the way, try also yo use a less concentrated gel. 15% is very high. Maybe your proteins are all stucked at the beginning because the gel is too concentrated. if you care resolving all the proteins, the best choice in would be to use a gradient gel 4-15% or similar. In this case, all the proteins will run and the smallest one will be retained in the gel as well.
As others mentioned, check the pH of the buffer used for making the gel. Prepare fresh buffer and check.
It looks like your mol wt markers ran in. Based on your previous work, did the markers close to 25 kD migrate to the distance you expected relative to the front? Knowing that could help eliminate some possibilities.If so, then your protein of interest might not have migrated into the gel because it precipitated out. Heat treatment with Laemmli sample buffer as suggested might work. Another step not mentioned is the use of reducing agent such as 2-mercaptoethanol when you prepared the sample for SDS-PAGE. That would help in case your protein has quaternary structure where 25 kD subunits are held together with disulfide bonds.
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