Results
Results for BSA (first week)
Concentration of BSA (mg/ml) / Absorbance at 280nm1.0 / 0.646
0.5 / 0.328
Results for Fractions( 1-4)
Fractions / Absorbance at 280nmF1 / 0.621
F2 / 0.504
F3 / 0.242
F4 / 0.440
The fractions F1-F4 were diluted at 1/50
Calibration curve were plotted from the BSA absorbance and concentration in order to find the concentration of these fractions 1-4.
BSA and fractions 1-4 calibrationcurve
Fraction / Concentration mg/mlF1 / 0.96
F2 / 0.78
F3 / 0.38
F4 / 0.68
Results for BSA (second week)
Concentration of BSA (mg/ml) / Absorbance @ 280nm1 / 0.628
0.5 / 0.311
Fractions / Absorbance at 280nm
F5 / 0.340
F5 diluted at 1/50
Calibration curve were plotted from the BSA absorbance and fraction 5 absorbance from the second weekin order to find the concentration of fraction 5.
BSA and fractions 1-4 calibration curve
Fractions / Concentration mg/mlF5 / 0.55
Section 1:
Present the results of your ouchterlony and describe which column fractions were pooled to form fraction 5 (F5)
Ouchterlony
Precipitation line was relatively visible for samples 2, 3 and 5.These samples were pooled to make fraction 5.
Section 2:
Calculate total protein of all your fractions F1-5. Present all values as:
- protein concentration in mg/ml
- Total protein in your sample having corrected your value for any dilution factor and the volume of the sample
A calibration curve must be completed and attached to this report. Sample values collected from the graph must be clearly labeled
Total Protein = Concentration (mg/ml) x dilutions x volume
F1: 0.96 x 50 x 0.5 = 24
F2: 0.78 x 50 x 0.5 = 19.5
F3: 0.38 x 50 x 0.5 = 9.5
F4: 0.68 x 50 x 1= 34
F5: 0.55 x 50 x 3 = 82.5
Fractions / Concentration (mg/ml) / Dilution / Volume (ml) / Total concentration (mg)F1 / 0.96 / 50 / 0.5 / 24
F2 / 0.78 / 50 / 0.5 / 19.5
F3 / 0.38 / 50 / 0.5 / 9.5
F4 / 0.68 / 50 / 1.0 / 34
F5 / 0.55 / 50 / 3.0 / 82.5
Section 3:
Calculate total IgG contained within all of your fractions F1-5. Present all values as:
- IgG concentration in mg/ml of each sample
- Total IgG in each sample having corrected your value for any dilution factor and the volume of the sample
A calibration curve must be completed and attached to this report. Sample values collected from the graph must be clearly labeled
1 0.5 0.2 0.1
At dilution 1/20, fractions F1, F2, F3, F5 circle formed. Fraction F4 didn’t form any circle.
Standards / Diameter (mm) / Diameter(mm2)1 / 13.32 / 177.4
0.5 / 11.21 / 125.7
0.2 / 8.90 / 79.21
0.1 / 7.11 / 50.55
Fractions / Diameter (mm) / Diameter (mm2)
F1 / 8.5 / 72.25
F2 / 8.8 / 77.44
F3 / 7.1 / 50.41
F4 / 0.0 / 0.00
F5 / 7.5 / 56.25
The diameter (mm2) for the IgG standard were plotted on a graph and the diameter (mm2) for each of the fractions that produced precipitation circles were plotted against the line of best fit to determine the IgG concentration in each fraction.
Single radial immunodiffusion of IgG
Calculate total IgG contained within all of your fractions F1-5. Present all values as:
Following calculation was used to calculate the total IgG in each fraction
Total IgG concentration = concentration x dilution x volume
F1: 0.20 x 20 x 0.5 = 2
F2: 0.23 x 20 x 0.5 =2.3
F3: 0.04 x 20 x 0.5 = 0.4
F4: didn’t form any circle.
F5: 0.08 x 20x 3 = 4.8
Concentration of IgG example of F1: look up diameter of circle which for F1 is 72.25 and look across line of best fit and go down which reads 0.20.
Fractions / Concentration of IgG / Dilution / Volume (ml) / Total IgG (mg)F1 / 0.20 / 20 / 0.5 / 2.0
F2 / 0.23 / 20 / 0.5 / 2.3
F3 / 0.04 / 20 / 0.5 / 0.4
F4 / 0.00 / 0 / 0.0 / 0.0
F5 / 0.08 / 20 / 3.0 / 4.8
Section 4:
Calculate percentage yield of IgG from the starting sample
((total IgG/ total protein) x 100)
F1 1:20 (2/24) x 100 = 8.3%
F2 1:20 (2.3/19.5) x 100 = 11.8%
F3 1:20 (0.4/9.5) x 100 = 4.21%
F5 1:100 (24/78) x 100 = 6.2%
Section 5:
Present the results of your immune-electrophoresis clearly labeling where your samples were loaded onto the gel and which antiserum was placed into which trough. Indicate where you observed lines of precipitation.
Plate 1
Fraction three with a-IgGproduced a single precipitation line, indicating no other proteins present and pure IgG is present.Fraction 1 with a-IgG produced multiple precipitation lines indicating fraction 1 contained other proteins. Therefore fraction 1 was not pure IgG. Moreover, fraction 2 with a-whs also produced multiple precipitation lines. lines indicating fraction 1 contained other proteins. Therefore fraction 1 was not pure IgG
Plate2
Fraction 3 with a-whs showed three precipitation lines so it’s not pure IgG. Fraction 4 showed no precipitin lines. Fraction 5 with a-whs showed many precipitation lines indicating clearly other proteins were present therefore, fraction 5 was relatively contaminated.