Urine Electrophoresis - mouse urine (Jan/28/2009 )
is someone out there familiar with electrophoresis of urine, especially urine from mice? can gels be evaluated like gels from human urinary proteins?
hope i would get some recommendations,
I've never done it, but if you can do it with samples from humans, you can do it with samples from mice.
The problem with the electrophoresis of urine is the concentration of the protein. Mostly you have the concetrate the urine to get a concentration of protein suitable for electroforesis.
It can be done bij concentrators or ultrafiltrationunits, whereby the ultrafiltration has the avantage that also the salt concentration can be lowered if needed.
After concentration it can be handeld as serum but be aware of the possibility that protein in urine can be degraded.
Non-concentrated human urine protein electrophoresis on agarose gel plate by myself modified
Gel agarose : a) Buffer: dissolve in 500 ml dH2O, 13.6 g/L (0.2 M) imidazole, 20.0 g/L (0.19 M) 2-amino-2-methyl- 1,3-propanediol, 58.0 g/L (0.94 M) boric acid, 0.4 g/L (1.07 mM) Na2-EDTA.2H2O, 1.0 g/L (3.47 mM) sodium dodecyl sulfate, and fill up to 1 liter with dH2O and mix. Aliquot and store at -20 °C; Agarose gel : melt 0.5 g agarose in 100 ml buffer and stirring in water bath at 70-80 °C until to agarose is completely dissolved.
Plate agarose gel: to position a glass that covers the whole surface of the superior edge of the water bath and to hold a temperature of the water to 50 °C so that the produced vapor maintains the temperature of the glass between 40 and 45 °C. On the surface of the glass a quadrangular space must be done with edges about 2 mm, in which to will find lodging an hydrophilic plastic sheet, on which, after acclimatization to 40-45 °C, the agarose fluid will be poured so that at the end a thickness between 0.5 and 2.0 mm has it. In each agarose gel plate few slots can be realized, not to all thickness, pre-loading slot, that can be loaded with about 5 µl urine sample.
Sample urine: 10 ml morning urine is collected, that is centrifuged at 1500 rpm for 5 min. and the proteins present in the sample were measured on supernatant with urine dipstick. If the proteine is greater than 2+, the urine samples has been diluted with saline solution to 1:1, and 5 µl of the sample dilution were dispensed in slot. Supply a tension <to 1.8 Á for 30 mins. Following electrophoresis run, the agarose films were immersed nigrosine stain (dissolve 0.05%