Advantages Of Dfr From Bl21 Ecoli Using Column Chromatography

The goal of this lab was to create and purify DHFR from BL21 ecoli using column chromatography. To know if we isolate DHFR their will be one band at 53 KD or above 50 KD and in our desalted eluate page should have the highestOD₂₈₀ which translates to the highest enzyme activity .Column chromatography is a method used to purify individual chemical compounds from mixtures of compounds. The main advantage of column chromatography is the relatively low-cost and disposability of the stationary phase used in the process.This process is used to manufacture prescription drugs such as for cancer
GST (glutathione s-transferase) which adds to increase solubility. DHFR (Dihydrofolate reductase), is an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid,required for synthesis,nucleotide,amino acids, DHFR inhibition or reduction disrupts nucleic acids synthesis affecting cell growth, propagation using NADPH as electron donor, which can converted to the kinds of tetrahydrofolate cofactors. In humans, the DHFR enzyme is encoded by the
Next we centrifuge to separate soluble from insoluble then we do affinity chromatography which the nickel sticks to the His-tag, after that eluate imidazole, nxt we do size exclusion which separates small from big,big comes off first the imidazole captures it in the column next we do spectrophotometry. Then SDS-PAGE electrophoresis, size purity,enzyme test does its function. Finally we did DHFR enzymatic assay. In the SDS-PAGE electrophoresis we load the gel up with Laemmli buffer, precision plus protein standard,desalted eluate page, eluate page, wash page, flowthrough page, soluble page, insoluble page, inducted page, and uninducted page. The desalted eluate page is the sample of interest since it should have the highest OD₂₈₀ and have the highest enzyme