Urgent...IEF voltage and current fluctuations

[sudamit]

Hi i am using a bio-rad Protean IEF Cell for the 1st dimension of 2-DGE. i am using plant sample and i am loading around 200 microgram of protein. after rehydration for about 16hrs when i start the 1st dimension their is a high fluctuation in voltage and current. the troubleshooting says it might be due to improper rehydration but the strip is getting completely rehydrated as is visible from the blue colour of the strip (7cm ph 3-10) imparted by the rehydration buffer. don't know why this is happening please help me out

[arnab_roy]

Hi Sudamit, I have done few 2DEs, but according to Bio-Rad manual we should not load more than 100 micrograms of total protein in a 7 cm IPG strip, rihgt? If your sample is a whole cell proteome then try loading 100 micrograms and if your sample is enriched in few hig habundant proteins then load less, may be 30 micrograms or less. Looking forward to your response.