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ANALIS

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

QUOTATION
INFORMATION
QUOTATION
INFORMATION
DESCRIPTION
DESCRIPTION

1. General

Date: 4 sep-03 - Revision: 01
EURO PN 844111002
PN 10-004060
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 protein specific stain for visual interpretation by comparing with a known reference pattern (control).

4. Content

Equilibration Buffer(1.33 mol/L TRIS-borate buffer) 1x200mL (PN 10-004061)

Electrophoresis Buffer (2.22mol/L TRIS-borate buffer) 1x200mL (PN 10-004062)

Protur Dye 1x100mL (PN 10-004064)

Agarose gels : 10x

Protur revealer (20 mL) 10-004063

Sample Templates - 10

Liquipette 2.5 mL

Measure (25mL) 1x

Template blotters 10 (19 x 126 mm)

Gel blotters 40 (83x101mm)

Drying blotters: 40 (76 x 101 mm)

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

- 10% acetic acid solution

- Saline solution

- Beckman Coulter's Paragon® Electrophoresis System

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 Equilibration Buffer: READY FOR USE.

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

Buffer is stable in closed container for 60 days.

Acetic acid solution 10%.

Protur Dye: 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.

Saline solution:

8.5 g NaCl in 1 L deionised water.

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: staining solution

· container 2: acetic acid solution

· container 3: acid alcohol solution

· container 4: acid alcohol solution

· container 5: deionised water

REUSABLE FOR 10 GELS

11. Sample preparation

Dilute urine when total protein is higher than 500mg/L.

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 5 µL

- Wait for 5 minutes

- Gently blot template with template blotter.

- Remove and discard blotter and template

Electrophoresis

- Pour 45 mL electrophoresis buffer in each compartment of the cell

- Locate gel on bridge

- Separate for 35 minutes at 100 volts

Detection

- Place gel in incubation box

- Distribute evenly 2 mL of Protur Revealer on top of the gel with a liquipette

- Incubate for 30 min. at room temperature

- Rinse in saline solution for 10 min.

- Place gel blotter on base of Press Dryer assembly

- Remove gel from gel frame and place gel, agarose side up, onto Gel Blotter on Press Dryer Base

- Place a second Gel Blotter moistened with saline onto gel surface, followed by 2 drying blotters and the press dryer top plate.

- Place the press dryer weight over the assembly and press dry gel for 10 min.

- Rinse in fresh saline solution for 10 min.

- press dry again following above method for 10 min.

- Place gel in oven (maximum 90°C) until dry

- Process gel as follows:

o staining solution 3 min.

o acetic acid solution 2 min.

o acid-alcohol solution 1 min.

o acid-alcohol solution 15 sec. (or longer until a clear background is obtained)

o deionised water 5 sec.

o Wipe excess solution

- Place gel in drying oven until dry (maximum 90°C)

- Evaluate gel

13. Calibration

Not applicable.

14. Analysis of results

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

1. Chopin N. Exploration biologique des protéinuries. Classification, méthodes d'investigation et interprétation. Spectra Biologie 1993;1:49-53
2. Chopin N, Le Carrer D. Etude de la sélectivité des protéinuries : description d'un nouvel index. Rev Fr Lab 1994;269:103-7
3. Chopin N, Le Carrer D. Exploration biologique des tubulopathies : mise à jour. Rev Fr Lab 1994;269:109-12
4. Le Carrer D, Nicolas A, Ducasse L. L'analyse des protéinuries au laboratoire de biologie en 1992. Rev Fr Lab 1992;245:41-7
5. Le Carrer D, Chopin N. Profil protéique urinaire : proposition d'un protocole d'exploration biologique des protéinuries. Rev Fr Lab 1994;269:29-37
6. 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
7. 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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