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EXERCISE 9: STANDARDIZATION OF TOTAL • Add 5.0 ml of biuret reagent to all the tubes
PROTEIN BY KINGSLEY • Mix well by finger tapping
• Stand for 30 mins at room temperature
OBJECTIVES • At the end of the 30 minutes period, read the
Identify the materials & equipment needed for the absorbance at 555 mu of tubes no.1 to 4 against tube
standardization of total protein no.5 (blank) using the 19 x 105 mm cuvette
Identify the parts of the refractometer • Convert the A reading to % T by using the conversion
Identify the purpose if the refractometer in Clinical Chemistry chart
Manipulate refractometer with utmost care
Enumerate the components of biuret reagent & state the GRAPHING & PLOTTING
purpose of each
State the principle of biuret method Ordinate graphing Absorbance vs Concentration
Identify a way of obtaining the concentration of an unknown paper
Discuss the procedure in making standard curve Transmittance % T readings vs Concentration
Differentiate the C-A from C-T graph graphing paper
State the importance of standard curve in clinical chemistry
→ this is your calibration or standardization curve, save this for
MATERIALS & EQUIPMENT the next experiment.
• Human Serum
METHOD OF OBTAINING PROTEIN
• Biuret reagent
CONCENTRATION
• Pipettes
• Spectrophotometer & cuvettes 1. In the total protein procedure (Kingsley), 0.5 ml of
• Refractometer serum is added with 20 ml of saline to give a total
• Pencil volume of diluted serum of 20.5 ml. Therefore:
• Ruler 0.5 𝑚𝑙 𝑜𝑓 𝑠𝑒𝑟𝑢𝑚 𝑢𝑠𝑒𝑑
= 0.0244
• Ordinate Graphing Paper 20.5 𝑚𝑙 𝑜𝑟 𝑡𝑜𝑡𝑎𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑑𝑖𝑙𝑢𝑡𝑒𝑑 𝑠𝑒𝑟𝑢𝑚
• Transmittance paper
2. In the total protein procedure, 5.0 ml of diluted serum
PROCEDURE is used. Therefore, 0.0244 x 5 = 0.122 ml of serum
actually used. This must be corrected to 100 ml, so that
PREPARE A 2.8 MG/ML PROTEIN STANDARD the result maybe expressed as gm/100 ml.
100
= 819.6 𝑜𝑟 820, 𝑤ℎ𝑖𝑐ℎ 𝑖𝑠 𝑡ℎ𝑒 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
• Get the total protein concentration of your serum by 0.122
using the refractometer
3. To determine what are the readings of the four
• Convert grams% protein to mg/ml protein
standards that should be plotted against: In gm/100 ml,
• Since only 25.0 ml TP standard is to be prepared, how
multiply the known amount of protein (2.8 mg = 0.0028
much volume (ml) _______ of serum is required?
gm) by the dilution factor of 8. Therefore:
• Let x represent the amount of standard to be used:
2.8 70
o = 100 0.0028 gm (1 ml) x 820 2.3 % TP
𝑥
o X= 4.0 ml of standard to be used 0.0028 gm (2 ml) x 820 2.3 % TP
• Pipet the calculated volume of human serum to be 0.0028 gm (3 ml) x 820 2.3 % TP
added by means of a volumetric pipet to a 25 ml of 0.0028 gm (4 ml) x 820 2.3 % TP
volumetric flask
• Dilute the volume with saline HOW TO USE THE REFRACTOMETER:
• Cover
• Mix well by inversion. This solution now contains 2.8 • Hold the instrument in a horizontal position if the stand
mg/ml of protein. is not provided. If a stand is provided, position it in such
a way that the position of the instrument is horizontal
INTO FOUR 16 X 50 MM TEST TUBE LABEL FROM • Lift the cover plate (A). Wipe dry with tissue paper.
ONE TO FIVE, ADD THE FOLLOWING: Wipe cover surface (B) dry with tissue paper
• Gently press cover surface A gently but firmly
Tube 1.0 ml of the prepared 4.0 ml of saline • With the use of a capillary pipet, place enough serum
1 protein standard to completely cover the whole prism or the cover plate
Tube 2.0 ml of the prepared 3.0 ml of saline A, as shown in figure 18
2 protein standard • Read Total Protein. Adjust & focus by rotating the
Tube 3.0 ml of the prepared 2.0 ml of saline eyepiece
3 protein standard • Record total protein in gm/100 ml
Tube 4.0 ml of the prepared 1.0 ml of saline
4 protein standard
Tube No addition of the prepared 5.0 ml of saline
5 CHON standard (label as
blank)
1
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