Hi !
I am trying to run some gels but everytime I do, my samples end up looking very wavy while the scale is ok.
For now my protocol is : sds page gel 12% acrylamide, run at 200V at room temperature for 40min. I prepare my sample by mixing them in a dye (commercial one) which I have to add beta-mercaptoethanol (BME) to it according to the notice. Then I put then 5 min at 95°C and I load them onto the gel.
I already tried to change few things in my protocol to improve my results but nothing worked I always have this huge blot in the end instead of thin strips. I changed :
- all the solutions and bought back every item so I am sure they are fresh.
- I ran the gel in the cold room at 4°C
- I ran the the gel at 120V
The only thing that comes to my mind now is to remove the BME because it is the only thing that I add compare to the scale.
Have someone had the same kind of issues ?
Thank you very much in advance ! :)
Jenny
Try running the gel at a lower voltage. I've seen that help improve band shape when there is trouble with it. If your sample is unusually viscous (for example, containing a high concentration of glycerol), that could be a contributing factor.
Are your initial samples in a buffer that is high in salt? I have seen similar artifacts occur for that reason as well.
Hi,
For gel preparation, ensure your acrylamide solution is properly polymerized before use. Besides, ensure your gel percentage aligns with your protein size.
For sample preparation, denaturing your proteins at 95°C for 5 minutes maybe be sufficient, but normally I denature proteins at 95°C for 10 minutes to make sure complete denaturation. And after denaturation, the sample should be vortexed to ensure no precipitation. Furthermore, it is essential to retain beta-mercaptoethanol in your loading sample. Instead, make sure that your samples are thoroughly mixed with the dye and beta-mercaptoethanol.
For running gel conditions, normally I start at 50 V for around 30 min, then increase to 90 V until the dye reaches to the bottom line of the gel glass plate (at room temperature). In addition, running buffer should be prepared correctly.
Also, amount of proteins shouldn't be loaded too much that may overflow the well.
I hope this information is useful for you.
Good luck!
Well-separated protein ladder indicates the running conditions, choice of gel, and general setup seem ok. Sharing your final Laemmnli sample buffer recipe would be beneficial to provide accurate recommendations.
You should pay particular attention to your sample composition. What do you have in your sample? Lipids, salts, urea...
It seems from the image you shared, that you loaded serial dilutions of the target sample but you obtained identical transitions. Therefore either sample buffer or sample ingredients must be problematic instead of SDS-PAGE set-up. Try to implement the traditional Laemmnli sample buffer recipe and if the same results are experienced, apply clean-up your sample prior to PAGE analysis (desalting, TCA/Acetone precipitation, ultrafiltration, etc...)
Good luck
- Badges
- Science method