HELP! PAGE Gel Running problem - (Oct/06/2006 )
Hi...
I am tryin to do PAGE... Using 12% separating and 4% stacking gel...
The problem i m havin is that, after Coomasie staining i see a completely blue lane in the lanes which have been loaded... And it seems that the running is only in the top half of the separating gel... Rest of the gel is clear... There r no visible bands at all... When i run with ladder, Then i can see the bands in that lane but that too only till the top half...
I am workin with cotton seeds... Want to do protein profiling of cotton seed storage proteins...
I am running at 100V in the stacking gel and then 200V till the dye front dissappears (Sometime an hour extra)...
Carryin out denaturation for 5 min in boiling water...
What can possibly be the problem????
I am tryin to do PAGE... Using 12% separating and 4% stacking gel...
The problem i m havin is that, after Coomasie staining i see a completely blue lane in the lanes which have been loaded... And it seems that the running is only in the top half of the separating gel... Rest of the gel is clear... There r no visible bands at all... When i run with ladder, Then i can see the bands in that lane but that too only till the top half...
I am workin with cotton seeds... Want to do protein profiling of cotton seed storage proteins...
I am running at 100V in the stacking gel and then 200V till the dye front dissappears (Sometime an hour extra)...
Carryin out denaturation for 5 min in boiling water...
What can possibly be the problem????
Oh dear. Well since your ladder isnt running, it seems like your gels are made incorrectly. Are they commercial or by yourself. Other possible reason is that it might be due to poor coomasie solution. Borrow someone elses would be a start.
have you checked the pH of your buffers?
are you using fresh APS?
are you giving the gels sufficient time to set?
Hi...
I am tryin to do PAGE... Using 12% separating and 4% stacking gel...
The problem i m havin is that, after Coomasie staining i see a completely blue lane in the lanes which have been loaded... And it seems that the running is only in the top half of the separating gel... Rest of the gel is clear... There r no visible bands at all... When i run with ladder, Then i can see the bands in that lane but that too only till the top half...
I am workin with cotton seeds... Want to do protein profiling of cotton seed storage proteins...
I am running at 100V in the stacking gel and then 200V till the dye front dissappears (Sometime an hour extra)...
Carryin out denaturation for 5 min in boiling water...
What can possibly be the problem????
Oh dear. Well since your ladder isnt running, it seems like your gels are made incorrectly. Are they commercial or by yourself. Other possible reason is that it might be due to poor coomasie solution. Borrow someone elses would be a start.
As i have told... Ladder is running but only till half length... the coomasie is also fresh...
Yes, it sounds like a buffer problem. It seems your gel is running normally until half way and then something.... is the buffer leaking into the gel box and running low? Is the buffer heating? Or it could be that the composition of the buffer is off. I would make fresh buffer or borrow some.
do you mean that you are running until the dye front runs off the gel? or does the dye front disappear?
if it is disappearing then you have to run the gel longer. i have seen the bromphenol blue dye front diffuse so that you can't see it (unless you put white paper behind and look very carefully) part way through the gel only to refocus near the bottom and then continue to run normally. if you allow it to refocus then your proteins will run as they should.
if you are running the dye off the gel, what is the range of your ladder, size of your protein. 12% is a relatively small pore gel and should be used for smaller proteins. larger proteins won't travel very far through the gel during the time of a normal run.
are you using fresh APS?
are you giving the gels sufficient time to set?
Well yes the gel is perfectly set and APS too is always freshly made...
I will recheck the buffer pH though... That could be the reson...
Will check and get back...
Thanx a lot...
I will recheck the buffer pH... That could be the reson...
Will check and get back...
Thanx a lot...
I am running at 100V in the stacking gel and then 200V till the dye front dissappears (Sometime an hour extra)...
do you mean that you are running until the dye front runs off the gel? or does the dye front disappear?
if it is disappearing then you have to run the gel longer. i have seen the bromphenol blue dye front diffuse so that you can't see it (unless you put white paper behind and look very carefully) part way through the gel only to refocus near the bottom and then continue to run normally. if you allow it to refocus then your proteins will run as they should.
if you are running the dye off the gel, what is the range of your ladder, size of your protein. 12% is a relatively small pore gel and should be used for smaller proteins. larger proteins won't travel very far through the gel during the time of a normal run.
The dye front does dissapear and is hard to notice... But i stop the runnin only after the dye front has run off the gel...
Cant understand why the complete colouration of the lanes and running only till half length or a bit less also sometimes???
Hi everyone...
I think i did find the problem... 
The pH of the tris base buffers was incorrect... Instead of 6.8 it was around 9 and the one which should be 8.8 was close to 13...
The readin on the pH meter still showed 6.8 & 8.8...
It was only when i checked with pH paper i found out the true pH...
Will make fresh solutions and try runin the gel...
Will get back to u ppl after that...
Thanx a lot all of u...