2D PAGE problem - (Nov/13/2006 )
We are currently experiencing problems with the second dimension of running 2D gels. We have always run the gels until the bromophenol blue (added to the agarose which is melted and poured over the IEF strip to hold it in place) is just about to drop off the bottom of the gel. This has always given us good separation of the proteins with haemoglobin (mouse liver) at the very bottom of the gel.
We are now finding that although we are running the bromophenol blue dye front to the same place, the proteins are not separating as much with the haemoglobin being only 2/3rds of the way down the gel (as opposed to the bottom). The dye front also has a “skewed” appearance as opposed to a straight line as seen in previous gels.
Our initial thoughts were that it was an acrylamide problem or a stacking gel buffer problem but these have been ruled out.
Does anyone have any ideas or experience with the same problem?
check your electrode buffer.
Checked that too. We were still using the same batch as we were prior to encountering this problem. We also made some new buffer incase and we still have the same problem.
Thanks
Anthea
Have you changed the gel percentage?
The dye front does not run at a particular molecular weight, but at a certain speed. So using two different gel percentages will cause the dye front to run at different relative speeds with reference to the protein in the gel. For the aparatus I use the dye front runs off at about 17kd in a 10% gel, but more like 10kd in a 12%.
As to the skewed front: you may be running at too high a voltage or for too long. If you use the biorad protean cell system you can run water through the aparatus to keep it cool.
We are now finding that although we are running the bromophenol blue dye front to the same place, the proteins are not separating as much with the haemoglobin being only 2/3rds of the way down the gel (as opposed to the bottom). The dye front also has a “skewed” appearance as opposed to a straight line as seen in previous gels.
Our initial thoughts were that it was an acrylamide problem or a stacking gel buffer problem but these have been ruled out.
Does anyone have any ideas or experience with the same problem?
Could be the voltage too high, but also salts too concentrated in your samples
The dye front does not run at a particular molecular weight, but at a certain speed. So using two different gel percentages will cause the dye front to run at different relative speeds with reference to the protein in the gel. For the aparatus I use the dye front runs off at about 17kd in a 10% gel, but more like 10kd in a 12%.
As to the skewed front: you may be running at too high a voltage or for too long. If you use the biorad protean cell system you can run water through the aparatus to keep it cool.
Thanks for the info jaknight.
Our initials thought was that it must be related to the gel percentage. We always use 12.5% and 3 people on seperate occassions had the same results which ruled out human error. We wondered if it was the batch of acrylamide so tried a new batch-we still had the same problem.
We do use the biorad system and cooling tank and we have always run the gels at 30mA per gel.
Still at a bit of a loss about all this. We are going to next try new APS and temed.
We are currently experiencing problems with the second dimension of running 2D gels. We have always run the gels until the bromophenol blue (added to the agarose which is melted and poured over the IEF strip to hold it in place) is just about to drop off the bottom of the gel. This has always given us good separation of the proteins with haemoglobin (mouse liver) at the very bottom of the gel.
We are now finding that although we are running the bromophenol blue dye front to the same place, the proteins are not separating as much with the haemoglobin being only 2/3rds of the way down the gel (as opposed to the bottom). The dye front also has a “skewed” appearance as opposed to a straight line as seen in previous gels.
Our initial thoughts were that it was an acrylamide problem or a stacking gel buffer problem but these have been ruled out.
Does anyone have any ideas or experience with the same problem?
Could be the voltage too high, but also salts too concentrated in your samples
Hi lillymay,
We are running at the same voltage as always (30 mA per gel) and we dont think it is a problem with salt as this would be evident in the first dimension. The spots have focused fine, they just arent far enough down the gel!
have you tried cleaning the electrodes in the gel tank?
what about the gel soaking solution? are you sure that you are denaturing properly? removing all the ampholytes? if your solution is too old or overused you may not be removing them properly.