You've got a lot of useful advice. I just can add a bit of physics to visualize the whole thing. The reason why macromolecules migrate in the gel is the difference of potentials (generally referred to as 'voltage'). The higher is the voltage the faster is the migration, and this relationship is linear. The voltage is the controling parameter here, the actual "driving force" of the whole process. On the contrary, the current depends on both the voltage and the resistance and tends to vary because the resistance increases gradually during electrophoresis. A bad thing about the current is that it consists of two components: one (the major) is provided by the small ions like Na+ or borate with which you supply your gels, the other (minor) reflects the velocity of macromolecules. You see, the trick is that the parameter you are really interested in (the speed of your macromolecules migration) contributes in only tiny part to the value of current, which depends much more on the buffer you use. Thus, by controlling the current you do not really control the proteins' or RNAs' migration. Conclusion: the fixed voltage is physically more appropriate in electrophoresis experiments.

Speaking about values, the rule of thumb developed ages ago is simple: use 10 to 15 V per cm of your gel. This gives about 100 V for small gels and up to 300 V for big ones.

The advice to set a top value for the current is still reasonable: overheating of the gel would be a much worse thing than an irregular migration speed.

Finally, as people have correctly pointed out, the beauty of your protein gels depends mostly on the concentration step. Use about twice lower voltages here to allow the samples stack on the border of the gels before you start the actual separation.

I am starting with 40 V at the beginning to let the samples line up. Then I am increasing the voltage to 100 V. The current is then around 115 mA, because the it increases proportionally with the voltage. So, there is a link between these two parameters. If you apply to much current, your gel could also melt.

Hi Mallu,

Some people use constant current for SDS-PAGE, but most use constant voltage. I run through the stacking gel at 50 or 60 V for 30 min. Then, the resolving gel can be run at 100 V or higher (I usually use 120 V or 150 V; some people go for 200 V if in a rush but this can give more diffuse bands).

It is important to keep an eye on the current, because as Dogan says, the gel can partially melt with too much heat. If you're seeing "warping" of the lanes, run at a lower voltage. As a rule-of-thumb, if the current in a minigel is getting significantly more than 50 mA, you should reduce the voltage.

The current will be higher at the start of the run, and gradually fall as the component proteins begin to separate. Let it settle down to a reasonably constant level over the first 10-15 min of the run, and check the current. If it's still too high, reduce the voltage.

That would be my advice, and I hope you find it helpful. The main thing to say is, just test out a few different protocols for running your samples and find what works best in the context of your project. Good luck...

Mark

Strictly depends upon the result you want, better resolution of bands at the stacking gel (if you are using the conventional gel casting technique) will enable a better resolution later on. If you use the currently available precast mixes i advice you to do this:

80 volts for 20 mins and 150 volts for 50 minutes. this will give good resolution.

I would strongly suggest you to play around with these values and see which is best suitable for your system

Unless specified differently I always run at constant voltage at 180V during the entire procedure. At this voltage I always get very sharp bands and no artefacts. When I run PhosTag gels though, I keep constant current ranging between 5-15 mA per gel. Voltage never rises above 150V but band focusing is suboptimal. This means that in this case the end justifies the means, so for routine applications, I would recommend constant voltage between 150-200V

I have the same doubt that you have. I Always run under constant voltage, 80 V, but the run extended around 3 hours. If I increase the run voltage, I have many problems in protein separation.

I have observed that constant current (15-20 mA) gives me sharp bands in low molecular weight range. Otherwise running at 80-100 volts or 15-20 mA hardly makes any difference.

You've got a lot of useful advice. I just can add a bit of physics to visualize the whole thing. The reason why macromolecules migrate in the gel is the difference of potentials (generally referred to as 'voltage'). The higher is the voltage the faster is the migration, and this relationship is linear. The voltage is the controling parameter here, the actual "driving force" of the whole process. On the contrary, the current depends on both the voltage and the resistance and tends to vary because the resistance increases gradually during electrophoresis. A bad thing about the current is that it consists of two components: one (the major) is provided by the small ions like Na+ or borate with which you supply your gels, the other (minor) reflects the velocity of macromolecules. You see, the trick is that the parameter you are really interested in (the speed of your macromolecules migration) contributes in only tiny part to the value of current, which depends much more on the buffer you use. Thus, by controlling the current you do not really control the proteins' or RNAs' migration. Conclusion: the fixed voltage is physically more appropriate in electrophoresis experiments.

Speaking about values, the rule of thumb developed ages ago is simple: use 10 to 15 V per cm of your gel. This gives about 100 V for small gels and up to 300 V for big ones.

The advice to set a top value for the current is still reasonable: overheating of the gel would be a much worse thing than an irregular migration speed.

Finally, as people have correctly pointed out, the beauty of your protein gels depends mostly on the concentration step. Use about twice lower voltages here to allow the samples stack on the border of the gels before you start the actual separation.

Very elegant answer from Alexander.

Want to add that you can also set the power constant in order to get a mix effect between warming and running at the same speed. Personally I prefer the constant amperage which is advice with my pre-casted gels.

By the way, for the transfer I always keep voltage constant because it provide me more reproducible transfer.

Thanks Alexander for explaining the physics behind it. Based on my personal experience, constant current generates less heat as compared to constant voltage. You can try running the smaller gel at 20mA in stacking and 40mA in resolving to get better resolved and sharp bands. Best.

Hi

We have not many samples to be run. So we usually run at constant current - at 30 mA/1 slap 1,5 mm SDS-PAG (traditional) as recommendation of PharmaciaBiotech, and so on, all results were quite good. But we thinhk you should take characteristics of the samples into acount. In our lab, we also usually do some preliminary experiments for new type of sample. Good luck

Hi Abhiram,

Better to prefer Constant Voltage.

Reference: Methods in Enzymology- Volume-463; Guide to Protein Purification, Academic Press-2nd Edn; 2009, Edited by Burgess R. Richard and Murray P. Deutscher.

I m using 80-110 volts cycle (In 4% and sep gel) for a mini-gel set up, but also check the temp of ur running buffer bottle-Running time will be short if it is close to RT and might be as longer as 3 hr or so if it is cold.

Gud Luck for your WBs.

It depends on the time available: Constant current guarantees a constant separation speed which is in most cases better to avoid the diffusion (and therefore diffuse bands).

Using constant voltage results in a decreasing separation speed the longer the electrophoresis is running.

So if you would like to run your gels (especially very long ones) overnight, use constant voltage. If you need to be much faster, use constant current, but here is a constant cooling important. Be aware: not colder than 15 °C, SDS will crystallize at lower temperatures.

If I am running 18% gel for the amyloid beta then what should be the conditions.

Please comment on this.

Both. In minigel, I do 100V 15min follow by 200V until gel ended or 40mA and 200V during all migration (around 1 to 2 hours). Both work well.

Dear Mallu

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.

However, in both methods, you must prevent the hotting of the runnig buffer to prevent damage to the proteins or gels

HI

For SDS- PAGE running the gel first you run at constant current mode of 10-15mA till you dye (Bromophenol blue) reaches the separating gel. Then you change to constant volt mode of 10-15V till the tracking dye reaches the bottom of the gel. Hence the separation and the supply from top to bottom be same. This the procedure i use to follow and the band separation resolution is also very good

Take a look:

http://www.bio-rad.com/webroot/web/html/lsr/support/lsr_electro_powerpac_supply.html

Dear Noorjahan

Using 60-80 V for stacking gel and 10-15 V per cm of the gel for separating gel (according to Sambrook), which is 100-150 V for larger gel works fine.

Alexandre's answer is explanatory and good enough.

Thank You Mallu for the discussion on this topic

I run with 200 V for 1 hr in ice or in cold room. Never faced any problem.

I hope this would work for you too.

The driving force of electrophoretic separation is the voltage (U). However, at the beginning of a run the resistance of the gel is low. If you were to use high voltage at this phase, current (I) would also be high and hence the power droped over the gel (P = U*I). This is turned into heat, and heat has several negative implications in electrophoresis. If you run the gel at constant current, the voltage will initially be low, but as the resistance of the gel increases during the run, voltage will increase gradually and eventually may even become limiting (when the power supply cannot provide enough voltage to drive the selected current). In other words, in constant current mode you try use as much voltage as the gel can handle without overheating. Some supplies have a constant power mode, which would be even better for this purpose.

Totally agree with Engelbert Buxbaum . Constant current mode ensures constant speed of separation .

Current represents the mobility of the ions including the protein-sds ions. Therefore, constant current would result in constant rate of migration. Also, constant current, makes the heat generated constant. Constant current results in predictable duration of the runs.