Immunology Lab Report

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  • Topic: Serum albumin, Absorption, Bovine serum albumin
  • Pages : 2 (321 words )
  • Download(s) : 243
  • Published : March 20, 2013
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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 technique.
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