I loaded approximately 20ug protein in each lane. After electrophoresis, the outermost lanes appear to have been spread outward rather than following a linear path. What could be the reasons behind. Picture attached for reference.
If the voltage is held constant throughout separation, the current and power (heat) decrease as the resistance increases. This results in longer but relatively cooler runs.
With current held constant during a run, the voltage and power of the gel chamber increase that results in shorter but hotter runs.
It appears that gel was running hot. I would check few things:
- The buffer composition.
- The age of buffer.
- The buffer temperature.
- Was there enough buffer in the tank?
- Power/running conditions (too high voltage?).
Hi! In my lab we run the gels in a cold chamber (8 ºC aprox), where we store the running buffer. This avoids excessive heating. Also, you can try setting the voltage instead of mA. Run it at 80 V for the stacking and up to 120-150 V for resolving. If your bands are too intense, dilute the sample.
Thanks for the suggestion! Is heating the only reason that leads to this situation? Because when I removed the gel and plates for staining the gel, I didn't feel the gel was heated. Do you think that when electrophoresed under constant current, may be the uniformity in flow of current across the gel is not maintained? What would be the difference if I run it in constant voltage instead of current?
Hi tias
You are supposed to run it at constant voltage b'cuz verticle elctrophoresis run at constant voltage and horizontal ones runs at constant current stack you samples in gel at 60V and resolve them at 80-120V if its a 10% gel. And at the time you cast your gel make sure the stacking line is straight and the running buffer you are using is fresh . As i can see from the image your gel seems to ruffles. You need to standerdise the sds composition in your running buffer II dnt see any problem with you pH8.8 tris hcl buffer cuz if there were any your bands wud be smeared
Thanks Sumit. But I want to know the conceptual reasoning for usage of constant current and voltage as you suggested. I have run all my SDS PAGE gels in constant current the way I have run this one, and never did I face any problem. It'll be helpful if you clarify me on that part.
If the voltage is held constant throughout separation, the current and power (heat) decrease as the resistance increases. This results in longer but relatively cooler runs.
With current held constant during a run, the voltage and power of the gel chamber increase that results in shorter but hotter runs.
Funny, we use constant voltage for horizontal electrophoresis and constant current for vertical one. Never heard of such distinction.
@Tias - I read just last week that it's better to use constant voltage because that makes more constant speed of migration. Something to do with the buffers and ions I believe, but I don't remember unfortunately. However, it was somewhere here on RG, so you may try to search for it.
Before loading sample gel should be submerge in to the buffer and second check applied voltage also.
The voltage is too high, you can start your run with high voltage until the samples passes out the stacking gel then lower the voltage for the rest of the run. good luck.
As far as I know, the intensity of the band is due to the amount of the protein in the well. The running voltage is only affect the protein separation rate
The trouble-shooting guide (link below) from BioRad might be of help in explaining Diffuse Bands and Lateral Band Spreading (in the section on Evaluation of Separation).
http://www.bio-rad.com/en-us/applications-technologies/performing-protein-electrophoresis#4
Bands will diffuse if the gel is left sitting once the voltage (run) has stopped.
The sharpness of the bands depends on many factors like the lysis process, choice of lysis buffer in itself, strange and the rate of seperation of protiens. Slower seperation rates leads to smeared and diffused bands.
I have the same problem now as I had to use the common Bio-Rad mini gels at room temp. We run the gels now in 2h or so with a bit cooling.
and there is always a broading at the bottom more or less. Before I used a gel chamber in a cool room and ran the gels very slowly over night. I never had this broadening and the gels looked just perfect with very sharp bands, so that i even could see the different glyco-forms of proteins. I think run time and temperature are serious points.
maybe you can also run the gel slowly over night and place the mini chamber in a fridge. or if you need 2h runs, precool the buffes, use cool packs in the reservoir, place the whole chamber on ice. try to avoid high osmolarities
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