Hello Asa, as usual in Biology there is no absolute rule that is valid for every case. But there are some principles which one could keep in mind, at least in a first approach.

In general, running at higher voltage/current (and this is true also for DNA agarose gels) gives better resolution for smaller size peptides (or DNA fragments). There are three reasons for this: smaller molecules are more subject to diffusion, both lateral and vertical, during the run, therefore in higher current the bands will be more compact and sharp, increasing resolution. But there is another factor influencing the run through the polyacrylamide mesh, the conformation of the protein. It is absolutely not true that in SDS loading buffer proteins are completely denatured (this is why the apparent MW on the WB seldom corresponds to the real MW of the peptide, and if you change running conditions, for example using MOPS or tricine gels, instead of the common tris/glycine gels, apparent MWs change substantially), but smaller proteins have less possible conformations than large ones, therefore there is less need to give to the peptides the time to find their lower energy conformation, and they will run more compact even with a fast run. Third, usually smaller proteins are separated on more concentrated polyacrylamide gels, the resistance created by this further increasing the heat generation, which is actually good if not excessive (and up to 150 V in a mini chamber at rt you are fine, but if you go to 300 V, as suggested above, you will have to cool), because it helps keeping the protein denatured, therefore reducing the possible structural conformations, which physically/sterically affect the run through the polyacrilamyde mesh, reducing the compactness of the band and, therefore, resolution.

Larger proteins, starting from 70-80 kDa as a rule of thumb, should be run at lower current/voltage (keep in mind that if you run at a fixed voltage the current will go down due to increase in resistance during the run, progressively slowing the run), because there is less diffusion and large proteins will assume more uniform conformation if you give them more time or find more uniformly their way through the mesh, typically at 110-120 V.

If the front of the gel smiles, that's a sign your gel is probably heating too much. Heat also swells the polyacrylamide gel, enlarging the pore size. This typically occurs mostly at the center because there heat dispersion is less efficient, making the center run faster.

As you say, a long stacking (at least 2 cm) run slow (80 V) will always highly improve resolution, because it will give time to the proteins to compact in a very thin front before starting the separation phase. But how well the proteins compact in the stacking, mostly if it is not long enough, will also be influenced by the amount of protein you load (with 40 ug of protein in a well from a 10-well minigel comb you will always obtain less resolution than with 4 ug... but then it depends if your proteins aresufficiently expressed to be detected, as well as if your Abs have enough affinity...) and by how much you fill the well: if you fit the loading in 10-15 ul you need shorter stacking run then if you load 30-35 ul, since the proteins are more diffused at the start in the latter case.

Also keep in mind that running gels at constant current will be faster, but heating of the gel will be less controlled, strongly and quickly increasing during the run due to the increased resistance, while running at constant voltage will slow down the run in time but will contain the increase of heat because you decrease the current flow over time... So, again, you might want to try the two depending on your protein...

Then, finally, you will always find cases where heating/cooling, running slow/fast, lower/higher pH is particularly beneficial or deleterious for resolution, you will have to find out yourself for your specific protein(s), case by case, if you are not satisfied of what you get...

Good luck!

Dear Asa

You are correct in assuming that the lower voltage is to control for heat as a result of resistance produced. It is important to consider both voltage and ampere though - I'm not sure what your set-up is but based on power-load in your building a lower voltage might not necessarily be associated with sufficiently reduced resistance, and subsequently heat production.

That said, running your gel in a cold-room, using pre-cooled buffer, would allow you to use a higher voltage. I'm assuming time is the issue here - you'd like faster electrophoresis times.

Good luck!

Regards,

Nathaniel

Lower voltage definitely helps in maintaining low heat. Having said that, you can run the gels even 300 V and get exact same results as you get at 100 V with the commercial gels available in market. It all depends on the quality of the material you are using to pour your gels, and to make buffers.

To separate close molecular weights you would need higher % gels or a gradient gel rather than voltage.

Hope it helps.

Good luck 

Venu

In addition to the lower heat you will generate in the system due to lower voltages, the lower voltage also helps in better resolution of proteins. If you are looking at proteins that are very different in molecular weight, you wont have any problems at higher voltages (as long as the system is cooled). If you are looking at proteins that are only 1 or 2kDa apart, then running the gels at lower voltages will help you resolve the individual bands better. 

Sanath may be right, but whats the science behind it, if not heat generation? So I agree with Nathaniel, except that a coldroom will not help, whereas precooled apparatus and buffers will. Keep these in ice, not a fridge. Some apparatus (BioRad miniprotein for e.g.)have an ice chamber for this purpose. But note that most apparatus have a thick plexiglass plate that effectively insulates the gel from the lower tank buffer: the upper (usually inner) chamber is much smaller and heats up very quickly (you can monitor that in real time using a thermocouple thermometer). When that starts to get too warm (I would guess 40oC) then stop the run, aspirate out the inner tank buffer and add more ice cold buffer.

Running SDS-PAGE in lower voltage results, as it was mentioned by Sanath, in more accurate protein separation. One of the reason of it might be not complete homogeneity through the gel, especially if it is self made gel. Passing not homogenous areas with low speed allows compensation of protein mobility differences. On the other hand, too long period of electrophoresis leads do increased band diffusion, although high temperature can increase diffusion as well. One should keep balance between time and resolution accuracy.   

Low voltage gives more time for protein to separate well on the gel, therefore we get better resolution at low voltage.At high voltage apart from heating proteins run very fast and dont get much time get separated very well.

Thank you all. You answers are such a good help.

Protein might have more time to separate if run slower, as Sanath and other mentioned. My advisor also said that. But what I think is that %acrylamide in gel forms polymer net or pore to capture different size of protein, so running speed might not be a factor to separate the protein. I think run slower run only during stacking gel is important for better resolution.

Why don't you do an experiment to determine the reality? I agree with you that PERHAPS a lower stacking gel voltage might help focussing, but comparing band resolution on one  chilled gel at high voltage versus one  non-chilled gel at low voltage might save you a lot of time in the future!! I might even do it myself!!

I was taught to run at constant current but the blog post below suggests otherwise:

https://www.researchgate.net/post/SDS_PAGE_should_be_run_at_constant_current_or_constant_voltage

The suggestion is to put an upper limit on your current to avoid heating. Best of luck.

In summary of the above nice discussion, lower voltage is good for several reasons:

1. to get good resolution of the bands, particularly to see the close bands

2. to avoid excessive heating as the buffer contains small ions particularly Na and borate receive heat

3. to maintain a standard velocity (running speed) of the macro-molecule in your protein sample during separation process

4. to avoid diffusion of your sample due to the excessive heating of the acrylamide

Best regards,

Hello Asa, as usual in Biology there is no absolute rule that is valid for every case. But there are some principles which one could keep in mind, at least in a first approach.

In general, running at higher voltage/current (and this is true also for DNA agarose gels) gives better resolution for smaller size peptides (or DNA fragments). There are three reasons for this: smaller molecules are more subject to diffusion, both lateral and vertical, during the run, therefore in higher current the bands will be more compact and sharp, increasing resolution. But there is another factor influencing the run through the polyacrylamide mesh, the conformation of the protein. It is absolutely not true that in SDS loading buffer proteins are completely denatured (this is why the apparent MW on the WB seldom corresponds to the real MW of the peptide, and if you change running conditions, for example using MOPS or tricine gels, instead of the common tris/glycine gels, apparent MWs change substantially), but smaller proteins have less possible conformations than large ones, therefore there is less need to give to the peptides the time to find their lower energy conformation, and they will run more compact even with a fast run. Third, usually smaller proteins are separated on more concentrated polyacrylamide gels, the resistance created by this further increasing the heat generation, which is actually good if not excessive (and up to 150 V in a mini chamber at rt you are fine, but if you go to 300 V, as suggested above, you will have to cool), because it helps keeping the protein denatured, therefore reducing the possible structural conformations, which physically/sterically affect the run through the polyacrilamyde mesh, reducing the compactness of the band and, therefore, resolution.

Larger proteins, starting from 70-80 kDa as a rule of thumb, should be run at lower current/voltage (keep in mind that if you run at a fixed voltage the current will go down due to increase in resistance during the run, progressively slowing the run), because there is less diffusion and large proteins will assume more uniform conformation if you give them more time or find more uniformly their way through the mesh, typically at 110-120 V.

If the front of the gel smiles, that's a sign your gel is probably heating too much. Heat also swells the polyacrylamide gel, enlarging the pore size. This typically occurs mostly at the center because there heat dispersion is less efficient, making the center run faster.

As you say, a long stacking (at least 2 cm) run slow (80 V) will always highly improve resolution, because it will give time to the proteins to compact in a very thin front before starting the separation phase. But how well the proteins compact in the stacking, mostly if it is not long enough, will also be influenced by the amount of protein you load (with 40 ug of protein in a well from a 10-well minigel comb you will always obtain less resolution than with 4 ug... but then it depends if your proteins aresufficiently expressed to be detected, as well as if your Abs have enough affinity...) and by how much you fill the well: if you fit the loading in 10-15 ul you need shorter stacking run then if you load 30-35 ul, since the proteins are more diffused at the start in the latter case.

Also keep in mind that running gels at constant current will be faster, but heating of the gel will be less controlled, strongly and quickly increasing during the run due to the increased resistance, while running at constant voltage will slow down the run in time but will contain the increase of heat because you decrease the current flow over time... So, again, you might want to try the two depending on your protein...

Then, finally, you will always find cases where heating/cooling, running slow/fast, lower/higher pH is particularly beneficial or deleterious for resolution, you will have to find out yourself for your specific protein(s), case by case, if you are not satisfied of what you get...

Good luck!

Thank you so much, Pietro. Your answer really enlightens me.