ProteoSpin™ Urine Protein Concentration Kits
For research use only and NOT intended for in vitro diagnostics.
ProteoSpin™ Urine Protein Concentration Kits
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Supporting Data
Figure 1. Two-Dimensional Gel Analysis. 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™ 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™ 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. 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 AlphaImagerTM 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.
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Kit Specifications
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| Maximum Urine Input Volume |
1 mL
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| Maximum Recovered Protein |
200 ÎĽg
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| Elution Volume |
100 ÎĽL
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| Time to Process 12 Samples |
20 minutes
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Storage Conditions
All solutions should be kept tightly sealed and stored at room temperature. Once opened, the solutions should be stored at 4°C. This kit is stable for 2 years after the date of shipment.
| Component | Cat. 17400 (25 preps) | Cat. 52300 (10 preps) | Cat. 21600 (4 preps) |
|---|---|---|---|
| Wash Solution C | 60 mL | 60 mL | 130 mL |
| Binding Buffer A | 4 mL | 4 mL | 8 mL |
| Elution Buffer C | 8 mL | 30 mL | 30 mL |
| Protein Neutralizer | 4 mL | 4 mL | 4 mL |
| Micro Spin Columns | 25 | - | - |
| Midi Spin Columns (assembled with collection tubes) | - | 10 | - |
| Maxi Spin Columns (assembled with collection tubes) | - | - | 4 |
| Collection Tubes | 25 | - | - |
| Elution Tubes (1.7 mL) | 25 | - | - |
| Midi Elution Tubes | - | 10 | - |
| Elution Tubes (50 mL) | - | - | 4 |
| Product Insert | 1 | 1 | 1 |
Documentation
FAQs
Micro, Midi, Maxi
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Centrifugation speed was too low.
Check the centrifuge to ensure that it is capable of generating 3,300 x g (micro kit), or 1,000 x g (midi and kits). Sufficient centrifugal force is required to move the liquid phase through the resin. Centrifugation speeds may be increased to 6,700 x g (micro kit), or 2,000 x g (midi and maxi kits), but this speed should not be exceeded.
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Inadequate spin time.
Spin additional 1 minute (micro kit), or 3 minutes (midi and maxi kits) to ensure that the liquid is able to flow completely through the column.
- Incorrect pH adjustment of urine sample.
Depending on a person’s acid-base status, the starting pH of the urine may range from 4.5 to 8. Therefore, it is important that the proper amount of Binding Buffer A be added to the urine sample in order to adjust the pH to 3.5 - 4 prior to loading onto the column. Especially with large volumes for midi and maxi kits, it is important to verify this and if necessary, add more Binding Buffer A to adjust the sample to pH 3.5 - 4.
- Initial volume of sample applied to column was too low.
Ensure that 1 mL (micro kit), or 5 mL (midi kit), or 20 mL (maxi kit) of the pH-adjusted urine sample is loaded onto the column in order to capture a large portion of the proteins present in the sample.
- Eluted protein solution was not neutralized.
Please refer to individual product manual for correct volume of Protein Neutralizer to be used in order to adjust the pH to neutral. Some proteins are sensitive to high pH, such as the Elution Buffer C at pH 12.5.
- Eluted protein solution was not neutralized quickly enough.
If eluted protein is not neutralized immediately, degradation will occur. We strongly suggest adding Protein Neutralizer to lower the pH.
