Team:UCLA/Notebook/Recombinant Expression/14 July 2015

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AGENDA

• BCA Assay
  • Complete BCA assay for acetone precipitation protein fractions from Monday's pooling and concentration of eluted fractions from Fraction #1 of the 1L Tamura outgrowth.
  • Add 1mL WR to each of the 13 samples, vortex gently to mix, and incubate at 37 degrees for 1 hour.
  • Remove and let samples cool to RT. Transfer samples into 1mL cuvettes.
  • Using the BCA Standard protocol in the spectrophotometer, measure both the blank, the standard samples, and the protein samples of interest using the machine readout.
  • Write down all absorbance values given and the predicted ug/mL concentration value given as well.

• IMAC Batch Purification of Fraction #2
  • Follow the protocol listed on last Friday for proper purification of the fraction.
    • Will use the equilibrate resin used last week for Tamura.

• Logistics
  • Order deoxycholic acid, lysozyme, imidazole, and protease inhibitors for the purification steps. Order through the Engineering requisition form.
  • Instruct J on downstream work until next Monday (in essence, preparation of a 1L Tamura batch that will be lysed, extracted, and purified at a higher concentration.
  • Continue brainstorming fundamental design tips.

RESULTS

• BCA Assay
  • Samples were blanked using 1mL of ultra pure water.

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

BSA Concentration (ug/mL)	Absorbance (562 nm)
2000	1.368
1500	0.147
1000	0.081
750	0.117
500	0.113
250	0.106
125	0.094
25	0.086
0	0.08

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

The resulting linear fit equation (y = 3E-07x+ 0.0877) 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 = 4)

Sample	Absorbance (562 nm)	Approximate Concentration (ug/mL)
3 (50 mM Wash Fraction)	0.092	143.333
4 (100 mM Wash Fraction)	0.093	146.6667
5 (250 mM Elution Fraction)	0.080	0
Concentrate (Pooled and Centrifuge Concentrated Fractions 3-5)	0.136	1610