It is dependent to the size of your gels. For bigger gels, I usually use low current and keep the electrophoretic tank in ice chamber. If your gels are small (such as Mini-protean of Biorad), it is not necessary to use this condition.

Using of low current is necessary only in the begging of PAGE running, when the protein are in the stacking gel.

My gel is small (mini-protean of biorad), I already run my protein sample at low current during first one hour, but the protein sample still migrate a bit further but not diffuse to the line of separating gel. So, I decide to run another one hour after that, then, protein samples and protein marker can reach the line of separating gel. But, i noticed that the migration rate of my protein sample and even protein marker was too slow, so i decide to run overnight by using low currrent and keep 120v constantly since i want to go home and next day i will observe the gel condition. Is it ok for me to do that?I already prepare fresh for all sds-page buffer, running buffer and even 10% APS. My MW of protein is just around 50 kDa. I used 12% separating gel and 4% stacking gel. I do not know which part that I did it wrongly. I get confused right now because i am not too experienced with sds-page. I am just a novice user and in-training graduate student.

How much current value do I need to set at initial part of running sds-page? How long to keep low current for waiting protein sample diffusing from the bottom of the gel to the downstream part of stacking gel before diffusing into upper part of separating gel?

you can change the % of the gel i.e in lower %

generally 20mA current in stacking and 18mA for separating (const current) (10% separating gel)

I often will run gels overnight with an OWL power supply and a mini-protean gel apparatus at 24V (typically about 5mA) on NextGel 12.5 and 15% gels. The voltage and current here are low and the buffer is different though so very little heat is generated.

In my experience the biggest problem with running gels overnight is heat buildup, if you can run your gel in a cold room this may help reduce the heat buildup (just remember to let your power supply warm back to room temp after before using it again to avoid condensation inside the unit).

I'd recommend doing a trial run with samples that are not essential and of know content. Theory and experience are great, but actual experimentation in this case can help you answer the question. Adapting and tweeking protocols to run overnight can be a big time saver for labs as you don't have to have people just waiting for things to run.

Of course you can run gel overnight, but I suggest checking concentration of stacking gel at first - it is a little bit strange that your protein as well as the marker is migrating so slowly in stacking gel. Are you using pre-casted gels or do you caste yourself?

You cannot run below the rate of diffusion of the bands that may be taking place in the gel. You may try 5-10mA per gel but not lower than that.

If you are using the commercially available gels from Biorad, you can use a higher voltage. Usually they can withstand voltage of up to 500 volts (check with the supplier). We have regularly ran gels at 200 V (run time-34 minutes) without any issues but since past 4-5 months we have increased the voltage to about 300 V and the run time has reduced to about 20 minutes.

Usually, 150-200V on BioRad should give you full separation of pre-stained markers (10-250 kD) on 10-12% acrylamide minigels in 1 h. If not, check all your buffers twice, and whole system for major leaks. With 50 kD protein you don't really need to run gel overnight under normal circumstances.

If you set your power supply to run at constant volts and run at 120 V, if your buffer solution is made correctly, you should be in the range of 20-35 mA for current, which you are not. Usually when this happens you are not completing the circuit - there may be a leak from your inner buffer chamber, so the buffer is below the level of your wells. Fill the inner buffer chamber and also the outer buffer chamber to the top so that if your inner buffer chamber is leaking it won't matter because the buffer will remain above the wells. You need the buffer to complete the circuit. Protein standards will completely separate in ~90 minutes or less at 120 V. If you run prestained standards you can monitor the run - if the protein standards separated, then the gel you poured or bought is good. Check your buffers to make sure they are the right strength, 1X SDS-PAGE running buffer and always use dI water to prepare your buffers. Also, when you put the gel into the gel box to make the inner buffer chamber - there is a green gasket on the inner buffer chamber that your gel presses against to make the inner buffer chamber. Your gel has a long glass plate and a shorter glass plate and the gel is inbetween these plates where you load your sample. The SHORTER glass plate should be facing toward the inside (for both gels if you are running 2 gels - if you are only running 1 gel, you still need either the plastic gel dam, or another plate to make the inner chamber), and the longer glass plate should be the one closer to you, with the shorter plate pressing on the green U shaped gasket. The gel presses against this gasket which is a little thicker on top to fill the gap in the short glass plate and helps to ensure your inner chamber isn't leaking. If you assemble this with the tall plate towards the gasket, your chamber will leak and you won't pull enough current at 120V for it to run.

the power supply depends on your gel size i.e. length, breadth, and thickness of the gel. when i was using biorad apparatus of size 11,10 cm and thickness 0.9 mm than 100 to 150 volt was sufficient to complete the run in 60 to 90 minutes.

when i was using LKB instrument with gell thickness 2mm then at 300 volt it took approx two hours. when i used the same LKB apparatus for 3 - 20% gradient gel then it took around eight hours to complete the gel. this i could left for overnight with slightly low voltage and complete the gel in ten hours.

if the migration is too slow then the expected then check the gel buffer, tank/ electrophoretic buffer and sample buffer for their strength, pH etc or prepare all of them fresh.

along with this you should also check the pore size of your stacking and separating gels are they appropriate.

This will be an enormous risk to take. trz precast gels 6-20%

I have run it extremely slow o/n and then in the morning, increased the voltage. For about 45 min, the maximum 150 volts for precast gels is used. I run at 10 volts o/n .

I agree that if it runs slow through the stacking gel, the pH of your buffer or % gel is off. It could be that even the loading dye buffer solution with sample is at the wrong pH or has too much salt, or glycerol, etc.