Immunology Lab Report
BRADFORD ASSAY
Calculation
Formulas:
Absorbance of BSA = A595 nm
Raw Data = Average of A595 nm of three wells ÷ 3 Example: A1+A2+A3 ÷ 3
In this case, 0.365+0.374+0.453 ÷ 3 = .397
Corrected Data =( (A595 nm well) – BackGround) or (Raw Data – Background)
Background = negative control = Bradford Reagent + No Protein
Background of this standard curve = A595 nm of well ‘A’ = .397
We used well ‘A’ as our negative control.
DATA
RAW DATA CORRECTED DATA BSA (µg) | A595 nm | 0 | .397 | 2.5 | .795 | 4.0 | .743 | 5.0 | .863 | 6.0 | .742 | 7.5 | .958 | 10.0 | .995 | BSA (µg) | A595 nm | 0 | 0 | 2.5 | .398 | 4.0 | .346 | 5.0 | .466 | 6.0 | .345 | 7.5 | .561 | 10.0 | .553 |
Standard Curve Raw Data (Figure 1)
Standard Curve-Corrected Data (Figure 2)
The figures, 1 and 2, show the relationship of Bovine Serum Albumin concentration and Absorption of light at 595 nm. Figure 1 shows the standard curve of Raw Data and figure 2 shows the standard curve- Corrected Data. Bovine Serum Albumin or BSA was weighed and dissolved in water to achieve a certain stock concentration. Water was used as a diluent. Quantitative dilutions of this stock were performed to achieve the indicated concentration, from 0.0 to 10.0 μg/μl, of BSA in a final volume of 25.0 μl. The concentration of BSA is shown in ‘X-axis’. The absorption of light by each concentration at a wavelength of 595 nm was determined with a spectrophotometer. The absorbance Values of each concentration is shown in ‘Y-axis’. From the both figures, we can notice that as the concentration level of BSA increases absorbance value at wavelength of 595 nm also increases.
In figures 1 and 2, we can see a decrease in absorbance value at BSA concentrations, 4.0 µg and 6.0 µg. This could be due to bad pipetting
Calculation
Formulas:
Absorbance of BSA = A595 nm
Raw Data = Average of A595 nm of three wells ÷ 3 Example: A1+A2+A3 ÷ 3
In this case, 0.365+0.374+0.453 ÷ 3 = .397
Corrected Data =( (A595 nm well) – BackGround) or (Raw Data – Background)
Background = negative control = Bradford Reagent + No Protein
Background of this standard curve = A595 nm of well ‘A’ = .397
We used well ‘A’ as our negative control.
DATA
RAW DATA CORRECTED DATA BSA (µg) | A595 nm | 0 | .397 | 2.5 | .795 | 4.0 | .743 | 5.0 | .863 | 6.0 | .742 | 7.5 | .958 | 10.0 | .995 | BSA (µg) | A595 nm | 0 | 0 | 2.5 | .398 | 4.0 | .346 | 5.0 | .466 | 6.0 | .345 | 7.5 | .561 | 10.0 | .553 |
Standard Curve Raw Data (Figure 1)
Standard Curve-Corrected Data (Figure 2)
The figures, 1 and 2, show the relationship of Bovine Serum Albumin concentration and Absorption of light at 595 nm. Figure 1 shows the standard curve of Raw Data and figure 2 shows the standard curve- Corrected Data. Bovine Serum Albumin or BSA was weighed and dissolved in water to achieve a certain stock concentration. Water was used as a diluent. Quantitative dilutions of this stock were performed to achieve the indicated concentration, from 0.0 to 10.0 μg/μl, of BSA in a final volume of 25.0 μl. The concentration of BSA is shown in ‘X-axis’. The absorption of light by each concentration at a wavelength of 595 nm was determined with a spectrophotometer. The absorbance Values of each concentration is shown in ‘Y-axis’. From the both figures, we can notice that as the concentration level of BSA increases absorbance value at wavelength of 595 nm also increases.
In figures 1 and 2, we can see a decrease in absorbance value at BSA concentrations, 4.0 µg and 6.0 µg. This could be due to bad pipetting