I running SDS-PAGE and I am facing a problem i.e. if I am setting voltage 100V then current is going to much higher like 400mA. If I am going through current and setting it 35 mA then voltage is automatically going 7V.
Gel is taking too much time to run, approx 9 hours.
Can anyone suggest me what should be the actual value of it, because at 400mA nothing is visual in Gel.
Dear Nilesh
There are no approved ratio between V and mA for PAGE.
It is better to constant current as 25-40 mA and provide cold condition (environment) for gel.
The consumed time is dependent to the length of gel and its thickness.
With 400 mA, your gel will be too warm and all proteins will be degraded and this can give more diffuse bands or no bounds. Also, the gel can melt with too much heat.
I run the stacking gel at 25 mA. Then, the resolving gel can be run at 40 mA.
Finally, as I said previously (https://www.researchgate.net/post/SDS_PAGE_should_be_run_at_constant_current_or_constant_voltage) Voltage is the electric potential difference between two points, or the difference in electric potential energy of a unit charge transported between two points. On the other hand, an electric current is a flow of electric charge. In electric circuits this charge is often carried by moving electrons in a wire. It can also be carried by ions in an electrolyte, or by both ions and electrons such as in a plasma.
Because may be differences between voltage and flow of electric charge that reach to proteins, it is better to constant current.
Hi Nilesh,
I share all the infos given by Masood in his previous post.
I always use ice cold buffer to prevent warming of my gel. It's a key tip!
I also use distinct voltage for my stacking and separating that gives me 20mA and 50mA. I have ever go through 100mA cause I was in a hurry and with cold buffer it was ok.
Maybe you can also check for the composition of the running buffer if everything is ok?
Hope you'll solve your trouble.
Thomas
based on my personal expereinces I started with 10 mA in staking gel while in separating gel i increase the power to 20 mA; and I have good results. My buffer kept always in 4 degree and I uae it directly from fridge.
Hi, It was also my concern for a long time a ratio between V and A.
In your case I think about problem with the buffers as Thomas said, check th pH, check the pH of your water. If your gels were handmade, check also the ph of the stacking and running gels solutions.
Perphas you have problem with your apparatus and power supply are they old or new? Do you control if there are residues of acrylamide on the electric wires of the apparatus, do you observe the classical bubble at the bottom, are they homogeneous?
It seems as you have resistance during the run, it is strange so you have to control the reagents and I would try to borrow another apparatus and power supply.
Good luck
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