STANDARD PROTEIN ASSAY-LOURY(MODIFIED)

STOCK SOLUTIONS
        SOL A) 20g Na2CO3 /L OF 0.1 N NaOH
        SOL B) 2% Na TARTRATE
        SOL C) 1% Cu.SO4.5H2O
        SOL D) 2N PHENOL REAGENT (FOLIN CIOCALTEU)

WORKING REAGENT (PREPERATION)

        1) BASE REAGENT                   TO INCREASE COLOR INTENSITY

                1ml OF SOL B                 2ml OF SOL B
              + 1ml OF SOL C                 2ml OF SOL C
                ------------                 ------------
                QS TO 100 ml WITH SOL A      QS TO 100ml

        2) PHENOL REAGENT (1N)

                1ml SOL D
              + 1ml dH2O
                ---------
                2ml 1N WORKING PHENOL REAGENT
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STANDARD CURVES

      -  REFERENCE STOCK = APPROX. 10g/dl (VALUE STATED ON BOTTLE)

      -  USING REFERENCE STOCK TO MAKE EXPANDED PROTEIN CURVE REQUIRES A 1/10
         DILUTION TO MAKE YOUR WORKING REFERENCE.
            EX. 1ml REFERENCE STOCK [10g/dL, 100 mg/ml] DILUTE TO 10ml TOTAL
                VOLUME WITH 0.9N NaCl. WORKING REFERENCE CONC. (10mg/ml)

      -  FOR PLASMA PROTEIN USE THE SIGMA PROTEIN STANDARDS ( 2g%, 4g%, 6g%,
                8g%, 10g%) STORED IN F-14 REFRIGERATOR.

      -  REMEMBER WHEN CONVERTING TERMS:
                MG% * 1000 = ug %
                ug% / 100 = ug/ml
         AND VICE-VERSA.

      -  ALWAYS USE A REFERENCE CONTROL WHEN ANALYZING IN EZPANDED CURVE.
         REFERENCE CONTROLS USED IN A-22 LAB HAVE THE FOLLOWING VALUES.
                         15mg/dl - 150 ug/ml
                         30 "       "
                         50 "       "
        *SLIGHT DIFFERENCES WIL BE NOTED BETWEEN ASSAYED VALUES OF HUMAN AND 
          BOVINE REFERENCES.
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METHOD
                   *FOR LOW CONCENTRATION SAMPLES*
A: 1) PIPET 100ul OF EACH SAMPLE INTO EACH 
        SAMPLE TUBE.
   2) PIPET 100ul OF EACH STANDARD INTO STANDARD TUBES.
   3) PIPET 100ul OF SALINE 0.9N NaCl INTO BLANK TUBE.

B: TO ALL TUBES ADD 2.0 ml WORKING BASE REAGENT, MIX THOROUGHLY.
C: WAIT 10 MIN.
D: ADD 100ul OF WORKING PHENOL REAGENT(1N) TO ALL TUBES , MIX THOROUGHLY.
E: TIME FOR 45 MINUTES (COLOR STABLE FOR 3 HOURS).
F: READ AT 650nm. O.D.
G: CALCULATE PROTEIN CONCENTRATION OF SAMPLES (O.D.) VERSUS CONCENTRATION OF 
        CONTROL VALUES (O.D.).
 
NOTE:
        THE MODIFICATIONS IN THIS METHOD WERE MADE TO FIT THE EQUIPMENT 
SPECIFICATIONS WE USE DURING ANALYSIS
        TO INCREASE  O.D. FOR EXPANDED CURVE A->D INCREASE SAMPLE, STANDARD
AND BLANK PIPETTED TO 200ul OR 300ul IN THE SAME 2.Oml BASE REAGENT VOLUME.