Figure 2.  Consistent Protein Purification from 1 mL Urine Samples.  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.

Figure 4. High Yield of Urine Proteins. Five different urine sample volumes were concentrated using Norgen’s Urine Protein Concentration Midi Kit. The total urine protein yield from the different urine sample volumes processed using Norgen’s kit appears to be increasing with the increase of the urine sample volume.

Figure 5. SDS-PAGE Gel Showing Total Proteins Purified from Different Urine Sample volumes using Norgen’s Urine Protein Concentration Midi Kit. Five different urine sample volumes were processed using the Urine Protein Concentration Midi Kit according to the bind, wash and elute procedure. The concentrated urinary proteins were eluted in the volumes recommended in the protocol based on the input amount, 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™ 2200 from AlphaInnotech. As it can be seen, total protein yield increases with the increase of the urine sample volume.

Figure 6. No Loss of Urine Proteins. The urinary proteins present in the 20 mL urine sample were concentrated into 2 mL of the provided Elution Buffer. The urinary proteins were resolved on a 12% SDS-PAGE gel run at 200 V for 75 minutes, and were stained used Commassie Blue staining. In the gel, Lane 1 contains 25 µL of input urine before concentration, Lane 2 contains 15 µL of the binding flowthrough, and Lane 3 contains 15 µL of the concentrated eluted protein. No urine proteins are lost during the binding step, as can be seen by examining the binding flowthrough.

Figure 7. Concentration of Urine Proteins. The urinary proteins present in 20 mL of urine were concentrated into 2 mL of Elution Buffer using Norgen’s Urine Protein Concentration Maxi Kit according to the provided bind, wash and elute procedure. The urinary proteins were resolved on a 12% SDS-PAGE gel run at 200 V for 75 minutes, and the urinary proteins were stained used Commassie Blue staining. Lane 1 contains 20 µL of the urine sample that was used as the input, Lane 2 contains 5 µL of the elution, Lane 3 contains 10 µL of the elution, and Lane 4 contains 20 µL of the elution. By comparing Lanes 1 and 4, which contain equal volume amounts of input and output, it can clearly be seen that the urine proteins have been highly concentrated though the use of the kit.