Why used as a protein concentration standard in lab experiments?
Answers
ABSTRACT:
The objective of this experiment is to determine the concentrations of RNase H which was purified in the previous lab experiment and of an unknown solution, which was administered by the TA. A Bradford reagent was utilized to determine the total concentration which binds to the protein. The samples were placed in a spectrophotometer and the absorbance was recorded for each sample. The data was compared to the calibration curve made using the standard protein solutions and the absorbance reading. Our sample was unknown #3, which had a total concentration of 1.418 mg/mL. The concentrations for the original flow through, washing buffer flow through, and the eluting buffer flow through were 0.021 mg/mL, 0.0274 mg/mL, and 0.014 mg/mL, respectively, with a 98% confidence interval of ± 0.0021 %.
INTRODUCTION:
In the previous lab experiment, the His-tag protein RNase H was purified by implementing a method called affinity chromatography. Affinity chromatography is utilized to isolate and purify proteins due to its high selectivity to the protein of interest (Biochemistry, 2015). The unused eluting buffer, original flow through, eluting buffer flow through, and the washing buffer flow through were all retained and stored for the latter experiment #7. In addition to the solutions mentioned, five standards protein solutions are prepared by diluting a 1.56 mg/mL of bovine gamma globulin solution (IgG) and unused eluting buffer. The final prepared solution is placed in a spectrophotometer and set at an absorbance of 596 nm. The unused eluting buffer is used as the control in the experiment to balance the spectrophotometer.