Figure 1.  Two-Dimensional (2D) Gel Analysis.  Urine protein samples purified using the Urine Protein Concentration Micro Kit are eligible for the analysis of protein profiles. Total urinary proteins were purified from 1 mL urine samples collected from a Hepatitis B virus infected patient as well as a normal healthy individual using this kit.  The protein samples were then separated on IPG strips (4-7) for 2D SDS-PAGE analysis. A total of 30 μg protein was loaded to the ReadyStrip^TM IPG strip pH 4-7 and focused on a PROTEAN IEF Cell at 250 V for 20 minutes, followed by 4000 V for 1 hour and then to 20,000 V. The strips were then transferred to the second dimension where they were loaded onto a 12% SDS-PAGE and run at 200 V for 75 minutes. The gels were stained using the Silver Stain Plus Kit from Bio-Rad and pictures were taken using an AlphaImager^TM 2200 from AlphaInnotech. The protein profiles of the Hepatitis B Virus infected patient and the normal individual (Control) are displayed above. The control 2D gel and the HBV 2D gel were analyzed and compared using Progenesis SameSpots software from Nonlinear Dynamics. The two gels were wrapped and overlaid to show the common spots as well as the difference between the two gels. The results suggested that this purification method is completely applicable for 2D protein analysis.

Figure 2. SDS-PAGE Gel Showing the Consistency of Total Proteins Purified from 1 mL Urine Samples using Norgen’s Urine Protein Concentration Micro Kit.   Seven different urine samples were collected, and 1 mL from each sample was processed using the Urine Protein Concentration Micro Kit according to the bind, wash and elute procedure. The concentrated urinary proteins were eluted in 100 µL of Elution Buffer, and 10 µL of each elution was resolved on a 12% SDS-PAGE gel and run at 200 V for 75 minutes. The gels were stained using Commassie Blue and pictures were taken using an AlphaImager^TM 2200 from AlphaInnotech. As it can be seen, consistent protein recovery was observed in each case.

Figure 3. Western Blot Showing the Recovery of Free PSA, ACT-bound PSA and a mixture of both from Spiked Urine Samples Detected using a Combination of Anti-free PSA Antibodies and Anti-ACT-bound PSA Antibodies.  Five milliliter urine samples were spiked with free-PSA, ACT-bound PSA and a combination of free-PSA and ACT-bound PSA. Total urinary proteins, including the spiked-PSA, were then purified using Norgen’s Urine Protein Concentration Kit. The urinary proteins were concentrated in 120 µL of Elution Buffer, and 12 µL of each elution was loaded onto a native 12% PAGE gel and run at 200 V for 75 minutes. Next, the proteins were transferred to a PVDF membrane using standard laboratory procedures. Once transferred, the membrane was probed using the Santa Cruz Anti-PSA antibody against free PSA and the Santa Cruz Anti-PSA/ACT antibody which is highly specific against ACT-bound PSA. The Sigma Anti-Mouse IgG-HRP antibody was used as the secondary antibody. The membrane was then developed using a Pierce CN/DAB Kit.  In the gel above, Lane 1 corresponds to free PSA, Lane 2 corresponds to ACT-bound PSA, Lane 3 corresponds to the free PSA and ACT-bound PSA mixture, Lane C corresponds to the input of both the free PSA and the ACT-bound PSA, and Lane M is the marker lane.