Catalog

17400

ProteoSpin Urine Protein Concentration Kit

( 25 preps )
Norgen Biotek

443.76$

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Additional description

Urine Protein Concentration Micro Kit (Cat# 17400 )

For the rapid and efficient concentration of total proteins from urine samples

  • Simultaneous concentration, desalting and buffer exchange of total urinary proteins
  • No molecular weight cutoff, therefore isolate all sizes of proteins and peptides
  • Process up to 12 samples in only 20 minutes
  • Also available in a 96-well plate format for high throughput applications

Product Description

Urinary proteins are important for many application such as Biomarker discovery, differential expression of proteins in various diseases, and diagnosis.The ProteoSpin™ Urine Protein Concentration Micro Kit is able to concentrate urine proteins several fold as well as remove salts, urea, and other urine contaminants. This kit has no molecular weight cut-off and therefore captures total urinary proteins and peptides of all sizes, making it ideal for biomarker discovery work or other diagnostic research work. This is an advantages over other commercial membrane devices and kitswith molecular weightcutoffs that limit the size ofthe purified proteins. The kit employs spin column technology that offers convenience, speed, and ease-of-use, and therefore offers significant time savingsover dialysis protocols.

The kit employsa proprietarymatrix whichacts as an ion exchanger and is able to concentrate proteinsmany fold as well as remove contaminants. Breifly, pH Binding Buffer is added to the protein sample and the sample is applied toNorgen's column (BIND). Under these conditions the proteins will bind to the column while salts and other contaminants are removed inthe flowthrough. The bound proteins are then washed to remove any remaining impurities(WASH). Lastly, thepurified urine proteins are elutedinto a small volume ofthe provided Elution Buffer(ELUTE).Please see theprocedure flowchart to theright.

Each spin column is able to concentrate and desalt up to 200 µg of urine proteins.The kit is also available in a 96-wellplate format for highthroughput applications.The resulting high-quality protein sample is concentrated and freefrom the original sample salts, thus preparing the sample conveniently for downstream applications including SDS-PAGE, 2D SDS-PAGE, Whole protein mass spectrometry, MALDI-TOF, LC/MS, LC/MS/MS, Western blotting and Protein microarrays.

FEATURES AND BENEFITS

  • Fast and easy processing - Process up to 12 samples in only 20 minutes, compared to other concentrating methods that takes a minimum of 35 minutes per sample. A 96-well plate can be processed in 60 minutes.
  • No molecular weight cutoff - Capture total proteins and peptides of all sizes, unlike other urine concentration methods with molecular weight cutoffs
  • Salt removal and buffer exchange - Norgen's kit allows for simultaneous salt removal and buffer exchange while concentrating urine proteins
  • Concentrated proteins can be used in a number of downstream applications - Final elution suitable forSDS-PAGE, 2D gel electrophoresis, Whole protein mass spectrometry and Protein microarrays

Specifications

Total urinary proteins and peptides purified usingNorgen's ProteospinTM Urine Protein Concentration MicroKit are concentrated and free from any original sample salts, and can be used directly in a number of downstream applications including:

  • Two-dimensional (2D) gel electrophoresis
  • SDS-PAGE
  • Whole protein mass spectrometry
  • MALDI-TOF
  • LC/MS
  • LC/MS/MS
  • Western blotting
  • Protein microarrays

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 ReadyStripTM IPG strip pH 4-7 and focused on aPROTEAN 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 wherethey wereloaded 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 andpictures were taken using an AlphaImagerTM 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 mLfrom eachsample 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 µLofeachelution was resolved on a 12% SDS-PAGE gel and run at 200 V for 75 minutes. The gels were stained using Commassie Blue andpictures 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 combinationof free-PSA and ACT-bound PSA. Total urinary proteins, includingthe spiked-PSA, were then purified using Norgen’s Urine Protein Concentration Kit. The urinary proteins were concentrated in 120 µLofElution 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 PSAandthe 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.

APPLICATIONS

Kit Specifications - Micro Spin Columns
Maximum Urine Input Volume
1 mL
Maximum Recovered Protein
200 µg
Minimum Elution Volume
30 µL
Time to Process 12 Samples
20 minutes

Kit Specifications - 96-Well Plates
Maximum Urine Input Volume
600 µL
Maximum Recovered Protein
120 µg
Minimum Elution Volume
50 µL
Time to Process 96 Samples
60 minutes


Urine Protein Concentration Kit Contents

1. pH Binding Buffer
2. Column Activation and Wash Buffer
3. Elution Buffer
4. Neutralizer
5. Micro Spin Columns (25)/ 96-Well Plate(2)
6. Elution Tubes(25) / Elution Plate(2)
7. Product Insert


Storage Conditions
All solutions should be kept tightly sealed and stored at room temperature. Once opened, the solution should be stored at 4°C.