This document summarizes the topics that will be covered in end of semester lectures, including coupled assays, buffers, the mechanism of AdhP, DNA sequencing, and a review of work on the AdhP gene. Specifically, it discusses considerations for running coupled assays, characteristics of good buffers, early experiments and a classic paper on alcohol dehydrogenase, basics of DNA sequencing using a simplified example, and the process of cloning the AdhP gene and purifying its gene product.

1. End of Semester Lectures
1) Coupled Assays
2) Buffers
3) Mechanism of AdhP
4) DNA Sequencing
5) AdhP Review

2. Coupled Assays
Anything that should be changed in the above assay?

3. “Stepping through” glycolysis forward and backward to
create possible coupled assays
What are some considerations when running coupled
assays?

4. Buffers: Choice of Buffer
The pKa of the buffer should be within 0.5 unit of the
desired pH (± 1 unit if you want to push it)
Potential interactions with a column matrix
Avoid UV-absorbing buffers if you plan to use a UV
detector
The ionic strength and salt composition must be chosen
according to the stability of the protein and the detergent

5. In other words, you want a “Good” buffer:
Hydrogen Ion Buffers for Biological Research*
Norman E. Good, G. Douglas Winget, Wilhelmina Winter,
Thomas N. Connolly, Seikichi Izawa, and Raizada M. M.
Singh Biochemistry, 1966, 5 (2), 467-477• DOI:

6. Biochemistry, 1966, 5 (2), 467-477

7. Biochemistry, 1966, 5 (2), 467-477

8. Biochemistry, 1966, 5 (2), 467-477

9. Biochemistry, 1966, 5 (2), 467-477

10. Preparation of Buffers
How would one make 1 L of a 2.0 M stock solution of
Tris·Cl at pH 8.0?

11. How would one make 1 L of a 1.0 M stock solution of
K+·MES at pH 6.5?

12. Mechanism of AdhP
1) Some of the early, crucial experiments on alcohol
dehydrogenase were performed by Frank Westheimer,
a physical-organic-chemist-turned-enzymologist
2) The following summary is from a classic 1953 paper
from the Westheimer Lab
3) If you are interested, Frank Westheimer also wrote a
very interesting article that appeared in the journal
Science (a very prestigious journal). The title of the
article is “Why Nature Chose Phosphates.” You can do
a Google search on the this title and get a PDF of the
article.

13. J. Biol. Chem., Jun 1953; 202: 687 - 697

14. H
H
from “Enzymatic Reaction Mechanisms” by Perry A. Frey and Adrian D. Hegeman

15. from “Enzymatic Reaction Mechanisms” by Perry A. Frey and Adrian D. Hegeman

16. from “Enzymatic Reaction Mechanisms” by Perry A. Frey and Adrian D. Hegeman

17. Some Basics of DNA Sequencing

19. Let us consider a very simplified example:
Let us say that our “gene” was the three-base
sequence: ATG
Since the beginning of any read is a bit noisy,
we want to start sequencing before the beginning
of our gene and beyond or after the end of our
gene. So, we will include the two bases upstream of
our gene and two bases downstream of the end of
our gene:
TTATGCA
The rest we will do on the board.

20. Review Slides of AdhP Work: from designing primers to
amplifying the adhP gene to cloning the adhP gene into
the pET101/D-TOPO vector to analyzing the vector to
transforming the vector into expression cells to using
affinity chromatography to purify the adhP gene product
(adhP)