Protein Electrophoresis. Technical Protocol

Size: px

Start display at page:

Download "Protein Electrophoresis. Technical Protocol"

Hugh Cross
7 years ago
Views:

1 Protein Electrophoresis Technical Protocol

2 Teacher: "What is the definition of a protein?' Student: "A protein is something that is made up of mean old acids."

3 Proteins are large molecules composed of covalently linked amino acids. Depending on electron distributions resulting from covalent or ionic bonding of structural subgroups, proteins can be either polar or nonpolar at a given ph.

4 Proteins were first identified and named by Jöns Jakob Berzelius in He and Gerardus Johannes Mulder stumbled upon them while studying substances found in animals

5 The separation of compounds based on their movement when exposed to an electric field was first observed in 1807 by Ferdinand Friedrich Reuß, who noticed the movement of clay particles in water when a constant electric field was applied. The theory of electrophoresis was refined in the early 1900s by Marian Smoluschowski, and further refined in 1937 by Arne Tiselius, who won the 1948 Nobel Prize in Chemistry for his work.

6 In the SPIFE SPE procedures, proteins are separated according to their respective electrical charges on agarose gel using both the electrophoretic and electroendosmotic forces present in the system. The proteins are then stained with a visible stain.

7 Menu Serum and Urine Protein Electrophoresis Serum and Urine Immunofixation

8 Specimen SPECIMEN: Minimum: 500 ul serum 10 ml urine Fresh serum or urine is the specimen of choice. Use of plasma will cause a fibrinogen band to appear as a distinct narrow band between the beta and gamma fractions.

9 Storage and Stability Storage and Stability: If storage of serum is necessary, samples may be stored covered at 15-30ºC for 4 days or 2-8ºC for 2 weeks, or -20ºC for 6 months. Urine samples may be stored covered at 2-8ºC for up to 72 hours or at -20ºC for 1 month.

10 Urine Concentration Urine Sample Preparation: Mix samples to homogenize. Centrifuge desired volume at 2000 x g for 5 minutes. Remove supernatant and concentrate as follows: Use Vivaspin 4 ml concentrators. Total Protein (mg/dl)conc. Factor <50 100x x x x >600 5x

12 Interfering Factors: Inaccurate results may be obtained on specimens left uncovered, due to evaporation. Hemolysis may cause false elevation in the alpha2 and beta fractions.

14 Helena Spife 3000

15 Electrophoresis chamber Sample cups and applicator blades on the left. Electrophoresis Gel on the right.

16 Sample Application/Electrophoresis Pipette the following amount of sample into the appropriate disposable cups: Serum or control: 45 ųl Concentrated urine: 20 ųl

17 Gel Placement Remove gel from packaging. Place the gel over the REP prep liquid in the chamber. Make sure there are no bubbles under the gel. Add electrodes on both sides of the gel.

18 Applicator Blades Remove applicator blades from packaging, making sure to use appropriate blade ( white for serum, gray for urine). Place blades into numbered vertical slots in the Applicator Assembly (spots 2, 8, and 14). For IFE place black blades in 4, 10 and 16.

19 Visualization After electrophoresis is complete, use the gel block remover to remove the gel blocks. Dispose of cups and blades. Place electrodes across ends of gel to prevent curling during drying. Close the lid and press TEST SELECT/CONTINUE button to dry the gel. After dried, carefully remove gel from electrophoresis chamber.

20 Visualization Remove gel holder from the stainer. Attach dried gel to holder and place in staining chamber. Select SERUM PROTEIN on display and press START button. The instrument will stain, destain and dry the gel. When the gel has completed the process, the instrument will beep. Remove gel and scan.

21 CALCULATIONS The Helena QuickScan 2000 densitometer will automatically calculate and print the relative percent and the absolute value of each band when the total protein is entered.

22 Sample Report

23 SELP Quality Control For Protein Electrophoresis two levels of controls are run daily with ranges established for all the protein fractions. Proteins fraction separations must be satisfactory.

24 Serum Protein Electrophoresis Stained Gel

25 IFE Quality Control IgG, IgM, IgA, Kappa and Lambda control serums are run daily to validate the antisera. Gels are evaluated for proper staining and separation. Patient samples with extremely high immunoglobulins are diluted and rerun to insure that no faint monoclonal bands are masked.

26 Serum Immunofixation Stained Gel

27 Interpretations Gels and scanned results are forwarded to the patholgists for interpretation.

Proteins. Protein Trivia. Optimizing electrophoresis

Proteins. Protein Trivia. Optimizing electrophoresis Proteins ELECTROPHORESIS Separation of a charged particle in an electric field Michael A. Pesce, Ph.D Department of Pathology New York-Presbyterian Hospital Columbia University Medical Center Rate of migration