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2.12: Total Serum Protein

  • Page ID
    120229
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    RELATED READINGS: Chapter 27. See Methods in CD-ROM for Total Serum Protein

    OBJECTIVES

    Upon completion of this exercise, appropriate discussion, and related reading, the student will be able to:

    1. Measure serum total protein with 90% accuracy using the biuret and refractive index methods.
    2. Compare the values obtained from colorimetric and refractive index measurement of protein.
    3. Identify any significant difference in values determined by each method.
    4. List the substance(s) that will interfere with each method.

    PRINCIPLE

    Biuret — Cupric ions in an alkaline solution will bind to the peptide bonds of protein molecules and form a blue-violet color. This complex can be measured photometrically and used to determine protein concentrations.

    Refractive Index - Serum or other fluids containing dissolved solids will refract incident light. The amount of refraction is proportional to the quantity of dissolved solids. If corrections are made for non-protein substances, this method provides a reliable estimation of protein concentration.

    glossary

    TS meter - another name for a refractometer. TS stands for Total Solids, which is what the refractometer measures.

    Normal saline -concentration of NaC1 (saline) approximating physiological concentrations, usually 0.15 M, 0.85%, or 0.9%.

    Reagent blank -a solution consisting of reagent and saline (instead of sample) in the proportions employed in the reactions used to “zero” a spectrophotometer (100% T).

    MATERIALS

    • Standard
    • Biuret reagent
    • Test Tubes
    • Controls
    • Distilled Water
    • Pipets
    • Patient samples
    • Spectrophotometers
    • 3% NAOH
    • Normal Saline TS (Total Solids) Meter

    PROCEDURE

    Biuret Method

    1. Label sufficient 13 x 100 mm test tubes for a reagent blank, controls, and the serum samples to be tested.
    2. Using a 5 mL serological pipet, pipet 4.9 mL of 3% NaOH into each tube.
    3. Pipet 100 \(\mu\)L of distilled water into the reagent blank.
    4. Pipet 100 \(\mu\)L of control, standard, or serum into each appropriately labeled test tube.
    5. Pipet 1.0 mL of biuret reagent into all tubes.
    6. Mix with a vortex mixer or by inversion.
    7. Incubate at room temperature for 20 minutes.
    8. Adjust the spectrophotometer to 520 nm and adjust for zero absorbance using the reagent blank. Transfer the reaction mixtures into an appropriate cuvette and measure the absorbance of each tube at 520 nm.
    9. Record your results on the data sheet and calculate the total protein values for each sample using the proportional calculation method.
    10. Record any unusual characteristics of the samples.

    Refractometer Method

    1. Clean the refractometer carefully with distilled water and blot dry.
    2. Place a drop of sample (standard, control, or serum) on the “stage” of the refractometer.
    3. Using sufficient light, look into the instrument and determine the total protein value from the appropriate scale and record it on the data sheet.
    4. Clean the “stage area” thoroughly with distilled water and blot dry.
    5. Repeat steps 2 through 4 for each additional sample to be measured.

    OPTIONAL ACTIVITY

    Measure the refractive index of distilled water, 0.85% NaCl, and any other material your instructor has prepared for you. Record your results on the data sheet.

    DATA SHEET, EXERCISE #12

    NAME: ___________

    DATE: ___________

    RESULTS

    Total Protein, g/L Absorbance at 520 nm Biuret TS meter Sample Comments
    Standard
    Normal Control
    Abnormal Control
    Sample #
    Sample #
    Sample #
    Sample #
    Distilled water
    0.85% NaCl
    Other

    Discussion Questions

    1. Were there any significant differences in the values determined by the two methods? If there were significant differences, explain the possible causes.
    2. Do you think that refractive index could be used as a regular method for serum protein determinations? If yes, what limitiations does it have. If not, why not?
    3. What are the major interferences in the biuret method? Refractometry method?

    2.12: Total Serum Protein is shared under a CC BY-NC-SA 4.0 license and was authored, remixed, and/or curated by LibreTexts.

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