Sandbox 323: Difference between revisions

Line 57:

The molecular weight of 3DS8 is proposed to be about 28 kDa.

In the laboratory, the gene was transformed into E. coli bacteria, expressed, and isolated by cell lysis and nickel column. The cell lysis buffer used was a sodium phosphate buffer. From the column, 10 elution fractions were collected. A Bradford assay with standards of 0.125, 0.25, 0.5, 0.75, and 1 mg/mL of Bovine Serum Albumin solution.

In the laboratory, the gene was transformed into ''E. coli'' bacteria, expressed, and isolated by cell lysis and nickel column. The cell lysis buffer used was a sodium phosphate buffer. From the column, 10 elution fractions were collected. A Bradford assay with standards of 0.125, 0.25, 0.5, 0.75, and 1 mg/mL of Bovine Serum Albumin solution. The standards were measured on a UV-Vis spectrophotometer at 595 nm. Initially, a few of the elution fraction were out of the standard range of absorbance, so the samples were remade by diluting further. After the second round of absorbances were obtained, only elution 4 was still out of range. Then Beer's law was used to determine the concentrations (mg/mL) of each elution fraction: E1=0.4026, E2=5.968, E3=5.434, E4=-0.1407, E5=0.5610, E6=0.4229, E7=0.03175, E8=0.1461, E9=0.2693, E10=0.2244.

Fraction E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure.

The ligand used for testing was p-nitrophenyl aldehyde in pH's of 4, 5, 6, 7, and 8. In each cuvette, 5 µL of protein, 50 µL of ligand, and 1.5 mL buffer (varying pH) were added. Absorbance was measured at 405 nm for 30 min, taking a reading every minute until 20 minutes, then every 20 seconds.

==Discussion==

Sandbox 323: Difference between revisions

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@@ Line 57: / Line 57: @@
 The molecular weight of 3DS8 is proposed to be about 28 kDa.
-In the laboratory, the gene was transformed into E. coli bacteria, expressed, and isolated by cell lysis and nickel column. The cell lysis buffer used was a sodium phosphate buffer. From the column, 10 elution fractions were collected. A Bradford assay with standards of 0.125, 0.25, 0.5, 0.75, and 1 mg/mL of Bovine Serum Albumin solution.
+In the laboratory, the gene was transformed into ''E. coli'' bacteria, expressed, and isolated by cell lysis and nickel column. The cell lysis buffer used was a sodium phosphate buffer. From the column, 10 elution fractions were collected. A Bradford assay with standards of 0.125, 0.25, 0.5, 0.75, and 1 mg/mL of Bovine Serum Albumin solution. The standards were measured on a UV-Vis spectrophotometer at 595 nm. Initially, a few of the elution fraction were out of the standard range of absorbance, so the samples were remade by diluting further. After the second round of absorbances were obtained, only elution 4 was still out of range. Then Beer's law was used to determine the concentrations (mg/mL) of each elution fraction: E1=0.4026, E2=5.968, E3=5.434, E4=-0.1407, E5=0.5610, E6=0.4229, E7=0.03175, E8=0.1461, E9=0.2693, E10=0.2244.
+Fraction E1 through E6 were chosen to be run on an SDS-PAGE gel because they contained the highest amounts of protein. After running for an hour on 100-150 mV, the gel was stained and destained. The bands appeared around 30 kDa and fractions E2-E6 were pure.
+The ligand used for testing was p-nitrophenyl aldehyde in pH's of 4, 5, 6, 7, and 8. In each cuvette, 5 µL of protein, 50 µL of ligand, and 1.5 mL buffer (varying pH) were added. Absorbance was measured at 405 nm for 30 min, taking a reading every minute until 20 minutes, then every 20 seconds.
 ==Discussion==