Postlab 2+3 - Postlab#2 Analysis of His-tagged Fusion...

Info icon This preview shows pages 1–2. Sign up to view the full content.

View Full Document Right Arrow Icon
Postlab #2 – Analysis of His-tagged Fusion Protein Expression in E. coli Introduction The purpose of this experiment is to analyze the overexpression of YqhD in E. coli through SDS-PAGE and immunoblotting to detect the His6 tag. It’s important to test overexpression at various time points to observe which time points the E. coli produce the most protein of interest. Two other conditions that can be changed to optimize protein induction are temperature and the concentration of inducer. This experiment uses mid-logarithmic phase cultures to induce protein expression because in the logarithmic phase is when bacteria are metabolizing and dividing. Lag phage bacteria are just getting use to their new environments and not metabolizing as efficiently, while stationary phase bacteria have slowed down their growth as resources become more limited. A gratuitous inducer is an analogue of a natural inducer that is capable of inducing an operon while not serving as a substrate for the enzyme that’s induced. This experiment employs isopropyl-thio-galacto- β-D-pyranoside (IPTG) as a gratuitous inducer for the lac promoter activity. This is advantageous as the gratuitous inducer will not be metabolized by the cells while the natural inducer for the lac operon, allactose, would be metabolized. The purpose of normalizing for cell density before loading the samples on the gel is to have the same number of cells loaded into each lane. Prestained standards are usually reserved for blotting experiments because they allow a quick way to determine whether the proteins migrated to the nitrocellulose correctly. Since the markers are prestained they can be seen no matter which detection method is used. The 20% methanol was added to the transfer buffer to promote binding of the proteins to the nitrocellulose membrane. The high concentrations of NaCl in the TTBS solution help by creating a high salt concentration which aids in protein immobilization on the nitrocellulose membrane. The reaction that leads to the formation of color
Image of page 1

Info icon This preview has intentionally blurred sections. Sign up to view the full version.

View Full Document Right Arrow Icon
Image of page 2
This is the end of the preview. Sign up to access the rest of the document.

{[ snackBarMessage ]}

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern