I have a technical problem with SDS-PAGE, the running sample is a protein extract dissolved in 100 mM NaCl solution, and others dissolved in 10mM PBS solution pH 8.0, the protein concentration per well is 8 ug and we used NuPAGE 10% bis-tris gel 1.0 mm, the running buffer was MES 1X, the running was set at 200 V for 35 min.
Try running the samples at a lower voltage for the first 10-15m till the sample mark enters the resolving gel. 200V is too high to begin with. I would start with 80-90V for 15 m and gradually go up to 100-110V until all the bands of the ladder are well separated. Also, check the running buffer concentration.
Good luck!
Also, try the most commonly used and effective protein extraction buffers:
https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-purification-isolation/cell-lysis-fractionation/cell-lysis-total-protein-extraction.html
These can be prepared in the lab easily and are also available commercially.
Thanks, Mukulika Bose for your answer, I will try that technique,
Thanks, Racheal Abuine
, also I will focus on this point.But the point here that, all of the samples have been reached the end of the gel but this pyramid shape formed because some of those samples couldn't go to the end of the gel and so they formed this shape, so my question what may make this happen??
I have seen this some times. We are using the same gels (NuPAGE and MES running buffer). Which type of tray are you using? I have seen it only on the new style tray where the two gels are mounted side-by-side - never on the older trays where the two gels share the top buffer chamber. It seems to help to fill the buffer chamber to the top even on the back of the gel.
Please try to run your gel at low voltage (~60-70 V) for a few minutes, say about 20-25 minutes, depending on the length of the gel. Then gradually increase the voltage to (~90-100 V).
I would recommend you run at low volt for optimization. Sometimes high voltage cause this problem in PAGE gel.
OR
Check for the electrode or its terminal for any kind of corrosion because the barrier in current flow is also one reason.
I think this a leakage stage of protein samples from the side of gel structure. It may refer to utlize higher voltage values that increased electric pressure inside gel. Thus, samples escaped from the sides and some of them scaped to running buffer. So, try to reduce voltage value.
Residual adhesive from the tape that covers the bottom of the gel could cause this.
I had a similar issue when having leftover magnetic beads in my protein extract. They somehow 'blocked' the pores in gel and lead to similar look. It also left this orange colour hue from the magnetic beads.
Thanks all Oliwia Mazur Christy Henson Mohamed Abdin Abhilek Kumar Nautiyal Bo Pontoppidan
All suggestions going to be checked and hopefully, this problem could be resolved.
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