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ANALIS

Protur™HiSi kit for Beckman Coulter Paragon® Electrophoresis - GB: Instruction for use

QUOTATION
INFORMATION
QUOTATION
INFORMATION
DESCRIPTION
DESCRIPTION

1. General

Date: 4 sep-03 - Revision: 01
EURO PN 844111009
PN 10-004100
100 tests per kit

figure 1


2. Intended use

The kit is intended for the electrophoretic separation of proteins in non concentrated urines.

3. Principle of the test

Proteins in an electric field migrate toward one of the electrode poles at varying rates. Migration separates the proteins. After migration the proteins are stained with a proteins-specific stain for visual interpretation by comparing with a known reference pattern (control).

A marker (internal standard) can be used for positioning of fractions.

4. Content

Equilibration Buffer(0.64 mol/L TRIS-borate buffer) 1x200mL (PN 10-004101)

Electrophoresis Buffer (3.2mol/L TRIS-borate buffer) 1x200mL (PN 10-004102)

Protur Dye 1x100mL (PN 10-004064)

Agarose gels : 1x10

Sample Templates - 1x10

Measure (25mL) 1x

Template blotters 1x 10 (19 x 126 mm)

Gel blotters 1x 20 (83x101mm)

When marker is used as internal standard: add 5 µL of I.S. (internal standard reference 10-004109) to 100 µL of urine.

5. Configuration of instrument

  • See Beckman Coulter's Paragon® Electrophoresis System.

6. Other reagent and material needed

  • Pipettes.
  • Graduated cylinder.
  • Deionised water.
  • Acid-alcohol solution
  • Saline solution
  • Beckman Coulter's Paragon® Electrophoresis System.

When using internal standard:

  • Marker (Internal standard) 1mL reference 10-004109

7. Training

The operator should be familiar with Beckman Coulter's Paragon Electrophoresis System.

8. Reagent preparation and storage

Agarose Gels: Just prior to use, carefully remove a Gel from the foil package. Gels should be stored at room temperature, 8°C to 30°C, until expiration date. Improper temperature or storage conditions can result in atypical migrations. DO NOT STORE GELS NEAR HEAT EMITTING DEVICES. For example: CRT, computer, or table top instrument. DO NOT REFRIGERATE OR FREEZE.

Protur HiSi Equilibration Buffer: READY FOR USE.

Protur HiSi Electrophoresis Buffer Mix 200mL of buffer with 800mL of deionised water.

Buffer is stable in closed container for 60 days.

Protur Staining Solution:

  • prepare acid-alcohol solution: 500 mL deionised water, 300 mL methanol, 200 mL acetic acid.
  • mix 100 mL Dye with 200 mL acid-alcohol solution.

Store reagents at room temperature

9. Type of specimen

Use fresh unconcentrated urine.

10. Instrument preparation

See Paragon® Electrophoresis System.

Place in wet processor:

  • container 1: dye solution (staining solution)

  • container 2: deionised water

  • container 3: acid alcohol solution

  • container 4: acid alcohol solution

  • container 5: deionised water

REUSABLE FOR 10 GELS

11. Sample preparation

.

No sample preparation needed.

When marker (Internal Standard is used): add 5 µL of I.S. (internal standard reference 10-004109) to 100 µL of urine.

12. Operations

Equilibration

  • Blot gel and pour 20 mL equilibration buffer in Gel Tray.
  • Place the gel, agarose down on the liquid.
  • Wait for 30 minutes (don't close the container)

Application

  • Blot gel
  • Locate sample template
  • Dispense sample: exactly 5 µL
  • Wait until whole sample has diffused in the gel.
  • Remove and discard template

Electrophoresis

  • Pour 45 mL electrophoresis buffer in each compartment of the cell
  • Locate gel on bridge
  • Separate 25 minutes at 100 volts

Staining

  • Process gel as follows:
  • staining (dye)solution 10 min.
  • deionised water 10 min.
  • acid-alcohol solution 1 min.
  • acid-alcohol solution 1 min. (or longer until a clear background is obtained)
  • Wipe excess solution
  • Place gel in drying oven until dry (maximum 90°C)
  • Evaluate gel

13. Calibration

Not applicable.

14. Analysis of results

figure 2

Evaluate gel by comparing it to a known reference pattern (control).

15. Calculation

Not applicable.

16. Imprecision

Not applicable.

17. Reference range

Not applicable.

18. Interference

None known.

19. Quality Control

Use a control sample.

20. References

    • Chopin N. Exploration biologique des protéinuries. Classification, méthodes d'investigation et interprétation. Spectra Biologie 1993;1:49-53
    • Chopin N, Le Carrer D. Etude de la sélectivité des protéinuries : description d'un nouvel index. Rev Fr Lab 1994;269:103-7
    • Chopin N, Le Carrer D. Exploration biologique des tubulopathies : mise à jour. Rev Fr Lab 1994;269:109-12
    • Le Carrer D, Nicolas A, Ducasse L. L'analyse des protéinuries au laboratoire de biologie en 1992. Rev Fr Lab 1992;245:41-7
    • Le Carrer D, Chopin N. Profil protéique urinaire : proposition d'un protocole d'exploration biologique des protéinuries. Rev Fr Lab 1994;269:29-37
    • Pourignaux F, Brohet M, Louis P, Chevigné R. News methods for detecting and identifying monoclonal and BJ proteins in urine without concentration. Sixth Int Symp of Nephrology, Montecatini Terme, Italy, June 1989
    • Van Hoof V, Chevigné R, Van Campenhout C, Lepoutre L. Evaluation of the Protur/Microprotur system  Beckman-Analis-Belgium for the protein electrophoresis of non-concentrated urine samples. 7ieme Coll Biologie Prospective, Pont à Mousson, 1988

Brochures:

B 25 E92 Analysis of Urinary Proteins by Electrophoresis without Concentration.

B 25a E97

B 46 D95 Urinproteinelektrophorese von nicht einkonzentrierten Urinproben.

B 38 F94 L'analyse des protéines urinaires par électrophorèse sans concentration.

B 39 E94 Urinary Proteins analysis by electrophoresis without concentration.

B 39a E96

B 45 D95 Elektrophoretische Urinproteinanalyse ohne Einkonzentrierung.

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