End of Semester Lectures

1) Coupled Assays

2) Buffers

3) Mechanism of AdhP

4) DNA Sequencing

5) AdhP Review

Coupled Assays

Anything that should be changed in the above assay?

“Stepping through” glycolysis forward and backward to

create possible coupled assays

What are some considerations when running coupled

assays?

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

Buffers: Choice of 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:

In other words, you want a “Good” buffer:

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

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

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

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

Preparation of Buffers

How would one make 1 L of a 2.0 M stock solution of

Tris·Cl at pH 8.0?

How would one make 1 L of a 1.0 M stock solution of

K+·MES at pH 6.5?

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.

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

H

H

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

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

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

Some Basics of DNA Sequencing

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.

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)