Team:UCLA/Notebook/Recombinant Expression/30 July 2015

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RESULTS OF BCA ASSAY

Table 1: Absorbance readings of BSA protein standards (n = 9)

BSA Concentration (ug/mL)	Absorbance (562 nm)
2000	1.874
1500	1.547
1000	1.122
750	0.883
500	0.586
250	0.341
125	0.199
25	0.095
0	0.075

Figure 1: Scatterplot of Standards fitted with a Linear Regression Line.

The resulting linear fit equation (y = 0.0009x+ 0.1111) was used to estimate the protein concentration of the unknown wash and elution fractions of the previous purification, as well as the concentrate formed using the pooled fractions described above, centrifuged in the protein concentrator. This equation was calculated where x is the protein concentration, and y is the absorbance value emitted by the spectrophotometer.

Table 2: Absorbance readings and estimated concentrations of samples (n = 12)

Sample	Absorbance (562 nm)	Approximate Concentration (ug/mL)
F1 (unbound protein)	0.920	796.8
F2 (20 mM Wash Fraction)	0.823	698.2
F3 (50 mM Wash Fraction)	0.762	637.5
F4 (100 mM Elution Fraction)	0.418	312.8
F5 (250 mM Elution Fraction)	0.118	50.20
Strip Fraction	0.078	16.39
C1 (Column Unbound Fraction)	0.438	331.0
C2 (Column 20 mM Wash Fraction)	0.090	26.52
C3 (Column 50 mM Wash Fraction)	0.190	111.7
C4 (Column 100 mM Elution Fraction)	0.090	26.52	C2 (Column 20 mM Wash Fraction)	0.090	26.52
C5 (Column 100 mM Elution Fraction)	0.092	28.17
CS (Column Strip Fraction)	0.080	18.04	-