I am using BIO-RAD PROTEAN II xi cell for protein gel electrophoresis. I am using 15mA constant current for first two hours then 35mA constant current for the rest of the run. My inner glass plate (16 x 20cm) always breaks from the middle into two pieces. I am cooling the system with cold water flow and not tightening the glass plate that much, but still every time I am have the same problem. Can anyone make any suggestions?
As I ran out of Bio-Rad plates, I used local glass plates of the same dimensions, but still had the problem.
Hi Parijat,
This is actually a mechanics and thermodynamics problem and not a biology problem. The breaking of the plate suggests that the conditions of the experiment are causing a stress to your plate, probably because there are uneven pressures applied to the system. One thing that you should absolutely be sure of are that your plates are cleaner than clean, even just a bit of dried gel from a previous run is a sure way to break your glass. Afterwards, assuming the pouring is ok and homogeneous, check thermic stresses as described by others and try to eliminate this for instance by running in a cold room and equilibrating the system before running. Also, your gel is found between 2 plates, are both breaking or is it just one of the two (inner or outer plate)? In case of just 1, check for mechanical or thermic stresses in that direction.
I hope this helps,
Jean-Marc
please only use voltage approximately 100-150 V until tracking dye 0,5 cm from bottom plate.
Voltage can never be a cause for this! attach the plates carefully in the instrument before the run.
How high is the voltage you apply here? This might be too high and cause cracking.
We always run at 35 mA constant current and haven't faced any problems yet!
I am attaching the plates carefully enough. Yesterday I ran my gel in 35mA , but the cracking was still thee, not from the middle but from a side.
I had a same problem, but I dont know how it worked for me. When I used apply wet spacer between the plate before casting I had a less breaking. It might help you too. I used to run at 10mA with stacking and then 20mA for separating. Keep track on voltage it should not go more than 200volt during run.
Good luck
Dhiraj
make sure that the plates are really clean, rinse them with distilled water before using, and also the device in which the gel is casted...
Can you run the gel in a cold room? or put the whole chamber into a bucket full of ice while it is running.
How many volts are 35 mA?
I had never expierence this problem, but glasses must be straight because bending can cause this problem
Hi Parijat,
We have seen this problem with a similar set up. The fact that it is the inner plate cracking would indicate too much heat. Even though you are cooling the apparatus, do you have any idea how hot the buffer is getting during the run? You may need to increase the water flow to help remove some of the heat from the buffer.
Hi Parijat,
This is actually a mechanics and thermodynamics problem and not a biology problem. The breaking of the plate suggests that the conditions of the experiment are causing a stress to your plate, probably because there are uneven pressures applied to the system. One thing that you should absolutely be sure of are that your plates are cleaner than clean, even just a bit of dried gel from a previous run is a sure way to break your glass. Afterwards, assuming the pouring is ok and homogeneous, check thermic stresses as described by others and try to eliminate this for instance by running in a cold room and equilibrating the system before running. Also, your gel is found between 2 plates, are both breaking or is it just one of the two (inner or outer plate)? In case of just 1, check for mechanical or thermic stresses in that direction.
I hope this helps,
Jean-Marc
I agree with Dhiraj. Its due to high voltage 1). try running your gel at lower voltage (lets say b/w 60-80 volts) till dye reaches at the junction of stacking and separating gels then increase it . 2). no matter u r using a cooling system , u cool the buffer in refrigerator prior to running of gel which could be replaced again after sometime with chilled buffer ( with some conventional method i mean u can use a 50 ml synringe for that). I think that would solve ur problem.
Its a principle of ohm's law, if you have more voltage, there is more resistance and this can lids to breakage of plate depending on the strength of plates. Thats why I have suggested voltage should not increase more than 200 volt. Alternatively you can prevent this by changing upper chamber buffer. As you are running at constant current, after some time ions from the buffer get depleted so automatically voltage goes up means there is more resistance.
good luck
Dhiraj
Sorry i am not using htis system and have never experienced such problem in other systems. My suggestion is change the system.
the plate should not be tightened enough when you cast. Also it should be very clean. When you add, TEMED, the buffer used to be hot. so don't pour it immediately but at the same time, it should not be allowed for sometime so that it should not get polymerised. Use minimum voltage say 100 V. Try to cast it in the airconditioned room. I hope this would be helpful to you.
As Dhiraj suggested, Try running the gel at constant voltage ( 80 V for stacking, 100 V for resolving) instead of constant current
It happens primarily due to high voltage. As you are saying that you do need to cool the system, it indicates that there is excessive heat generated. Try running at constant voltage 80 to 100 and it should not happen. good luck
Hello
It happened to us as well with the same system
The glasses are super-clean, tighten just as much as needed, we follow the voltage instruction as reported on the guidline…. The inner plate always brakes down (sometimes also in the cold room).
The only way it works for us is to run it at constant voltage overnight in a cold-room.
If I remember well we let the gel run between 6-8 mA
however I am very disappointed wit this. The old fashion apparatus (same size same brand) has never given a problem. The new one seems to fall into pieces
Michela
Although,i used same BioRad system, i/we never experience such breakage. I guess, you need to more cautious right from preparation and running of gel. or the plates obtained might be quite fragile. Also,heat generates due to prolong running of system at higher voltage. As previously, suggested you can try running initially at 50-60 volts,urther you can increase the voltage accordingly tll 100 volts. Best luck.
The best way to run is at constant Wattage, since that determines the heating effect. You should be able to run at 25W with little problem, even higher if you are in a cold room, or have cooling water running through the system.
Hi Parijat, as Jean -Marc has suggested, it could be the reason of uneven pressure on your plates. While making cassette for the gel use both hands to tight bolts for right and left side, and, to have even pressure all over the plate you have to work on all bolts same time, I mean don’t do full tight at one place first then go other side. Give turn to all bolts little bit little bit. This is for Amersham system what I have in the lab; I am not sure which kind of Bio-Rad has. You can also contact their tech. By the way if you are using clamps (the old fashion), pick the clamps which ones feel even strength.
Decrease ur current.. Dont go beyond 15 mA it will be all fine. Its typical heating problem and use fresh buffer everytime you run ur gel
Hi,
I agree with keeping the voltage to 80-100 V, preferably in a cold room. I filled the chamber with anti-freeze/cooler, the one you normally use for your car. It works way better than water, and less mess. Good luck! Olga
Dear Prijat,
try to see the glass plate holding cassette there may some distrorsion in the holder which will exert uneven pressure on the glass plate and cause to crack them. i also have face such problem with my biorad mini gel system. the plastic module to hold the plate was slightly distorted by pressure and that exert uneven pressure on the plates.
Hi prijat,
You said used local glass plates of the same dimensions, do not use local glass plates The best way to use biorad system glass plates only, because we also face same problem with GE health care system glass plates then we gone for biorad system maintain voltage 75-100V, good luck
The bio rad glass is very thin, we replaced all our systems with atto, don't remember the last broken plate that was as a result of anything other than idiocy
simple..., after clean, never dry the glass in the incubator, always at room temp, works for me for Biorad glass plates.
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