help with LDH separation using agarose gel electrophoresis - (Aug/03/2007 )

Hi all,
I'm currently trying to optimise a protocol for separating LDH isoforms using agarose gel electrophoresis, and have so far had no success. I've tried using 200-250v, 1-2% gels, for 1-2 hours, and am running the gel on ice to cool the tank (appleton woods horizontal agarose gel electrophoresis system).
Despite the ice/dry ice mixture, the gel starts to melt, as the tank reaches >60 degrees C. The next thing i'm going to try is lowering the voltage (100v) and agarose% (0.5%) and running it for longer (4 hours), however I'm losing faith because so far the bands have neither separated, nor run further than a few centimetres.
Has anyone got any advice/ had any experience with this technique?


ps I'm visualising the LDH with a TBZT+PMS+NAD reagent.

What buffer & pH system are you using? One technique that might help is to overlay the gel with a reaction mix made up of 500mmol/l lactate with 13mmol/l NAD. Incubate at 37degs to genarate NADH and detect at 340nm.

There is another way of using differential heat stabilities in comparison with total activity at standard temp. You incubate at 57degs for 30mins and at 65degs for 30mins. The stable fraction left after heating at 65degs is usually 20to 40%, rising to 45to65% in myocardial infarct. The heat labile fraction 9which you heated off at 57degs) is 10to25% rising to 33to80% in liver disease.

Last idea, use cellulose acetate electrophoresis.

cheers guys, i'm going to have a go with an acrylamide gel today, but i'm also looking into getting an assay kit from Helena biosciences- any ideas whether this is a good move? anyone had any experience with these guys?

We use the Helena ALP iso enzyme kit. Its straightforward and reproducible.