2D PAGE problem

[antheab]

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?

[moleculardude]

Hey there antheab and welcome here!

You could have quite a few problems so maybe we can narrow them down.

Buffer Problems

Your Running buffer (for running gels) may be the source of your problem (pH or salt problem)

Your lysis buffer (new source of tissue? Cells?) may be the problem due to changes in salt or lysis conditions.

Are you sure you are using the right percentage of polyacrilamide gel? (maybe you are running higher percentage by accident?

Gel Running Problems

Did you check your voltage? current? Is it the same as before?

IEF / Agarose

Last but not least, your agarose and/or IEF buffers may be affecting the proteins and the way they run.

Sorry for the long list. Just narrow it down!
Please post back here so we can continue helping you and find out what solved your problem... dont leave us in agony!

[antheab]

Thanks for all your suggestions moleculardude.

We know its not the running buffer as we are still on the same batch as prior to the problem. We also tried some fresh.

We are confident it isnt the acrylamide. We have also tried a new batch.

The voltage and current is the same as always.

We didnt think it was a problem with the IEF buffers as the focusing looked fine.

In what way do you think the agarose might be causing a problem?

Thanks
Anthea

[bojanab]

Hi Anthea,

I am experiencing the same problem you had (your post from 2006):

Originally Posted by antheab

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?

I work on plant proteins and always had a good separation of these proteins but then this problem started to happen (proteins are stucked in 1/3rd of the gel).

The problem is not caused by AA, TGB, current, voltage, IEF buffer or equilibration buffer.

Did you reach any conclusion regarding this problem? It would help me a lot