Will you process the sample by 1D SDS-PAGE LC-MS/MS? If you want identify different proteins as much as possible, you may load a more sample amount in 1D SDS-PAFGE. If you want identify the protein spots from 2DE, MALDI-TOF MS might be a better choice.
About your program, how much is the electric current at 10000V, when the step finished. And I suggest 8000 V or 6000 V for 13cm strip. dye have not reached the (+) end, it may indicate the sample is impurities (containing fat, polysaccharide et al. ). The Clean-Up Kit (GE) or TCA- Acetone precipitation may be helpful to sample purification.
Bile fluid samples were sonicated and centrifuged at 16 000 g for 15 minutes at 4 ℃ to remove debris, nucleic acid and mucins as a preliminary separation. For sample delipidation, each 1 ml of preliminary separated sample was then mixed with 250 µl of CleanasciteTM HC (Ligo-Chem, Inc., Fairfield, NJ, USA) followed by rotation for 1 hour at 4 ℃. After incubation, each sample was centrifuged at 16 000 g for 1 minute to clear away the formed lipid-micelles, and the supernatant was transferred to a new tube. In order to reduce the salt concentration and other contaminants, a commercially available microcentrifuge filtration device (YM-3, molecular mass cut-off at 3 kD; Millipore, Bedford, MA, USA) was used to wash away contaminating species.
There is not any problem to using the Kit for protein quantification.
How do you prepare your sample？
do the proteins precipitated and dissolved in lysis buffer (Urea buffer)? If not, it will make bad IEF results, because of salt, fat, saccharide… in samples.
I quantify proteins using 2D Quant-Kit (GE Healthcare). Bradford has a bad compatibility with Urea, Thiourea and DTT. So I recommend 2D Quant-Kit for quantification of proteins dissolved in Urea buffer.
What was the length of the IPG strip you used?
I think you can increase the amount of loading samples. GE Healthcare Handbook just is a recommendation. The amount of loading is based on the type of sample (from cells, tissue, membrane proteins, nuclear proteins and so on). If your sample is complexity (including thousands of different proteins) you should increase your loading amount. Furthermore, the length and pH rang of the IPG strip is the factor you should consider. pH 3-10 maybe need a high sample amount.
Samples from tissue 250 ug for 24cm pH 3-10 IPG strip (silver strain)
180 ug for 18cm pH 3-10 IPG strip (silver strain)