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Lab #8: Kinetics and Sequencing

March 8 & 10, 2015

Hot Tip for Today

Write down all quantities and
concentrations; youll need them later.

even from your stocks!

Enzyme Kinetics Basics

Rate order
Dependence of reaction rate on number of species
reacting
Zero order: rate does not depend on [S]
First order:
A P
Rate = k [A]

Second order:
A + B P + Q
Rate = k [A] [B]
LDH mechanism*

Enzyme Kinetics Basics

NAD+

lactate

E NAD+

E NAD+ lactate

pyruvate

fast

E NADH pyruvate

Simplify: How????

E NADH

NADH

Michaelis-Menton Kinetics
Rate (umol/min)

Vmax

Vmax

KM

[S] (uM)

Lineweaver-Burke Plot
**Introduces lots of error in
KM and Vmax

Competitive Inhibition

Increasing inhibitor

Affects KM
Affects Vmax

Competitive Inhibition

Increasing inhibitor

Affects KM
Affects Vmax

Uncompetitive Inhibition

Increasing inhibitor

Affects KM
Affects Vmax

Uncompetitive Inhibition

Affects KM
Affects Vmax

Non-Competitive Inhibition

Affects KM
Affects Vmax

Non-Competitive Inhibition

Affects KM
Affects Vmax

Increasing inhibitor

Use the Lineweaver-Burke

to determine inhibition type
Y-intercept: 1/Vmax
X-intercept: -1/KM

Competitive

Noncompetitive

Uncompetitive

Eadie-Hofstee
I recommend that you read about it on Wikipedia

y = m x + b

Be comfortable reading, interpreting, knowing the

equations for, and creating all 3 types of kinetic plots!

Michaelis-Menton Kinetics

Lineweaver-Burke Plot

Eadie-Hofstee

LDH Kinetics
Testing kinetics of concentrated SEC/AFC LDH
sample
Determine which has better activity
Determine sample volume that gives:
Abs/min ~ 0.15-0.25

Kinetics:
Varying [NAD+] to plot velocity vs. [NAD+]
Can determine KM for NAD+ and Vmax of LDH

Follow instructions very carefully

Record everything!

Sequencing pET15b/LDH DNA

First thing: Plasmid prep of DNA
Is the LDH gene insert inside pET15bs MCS??
2 ways to check:
1. Digest plasmid with NdeI and BamHI REs and run
it on a gel looking for
2. Sanger sequencing

#1: Digest DNA with REs

A
DH A
KG
L
r
/
/
H
H
T
T
G
p E
/LD
/LD ig pE T/rK
g
T
T
r
i
E
e
pE ig pE
t D ig p
t D
dd
La
Dig
D
No
D
No

Digest
NdeI & BamHI

5 kb

2 kb
1 kb

#2: Sanger Sequencing

If your digestion didnt work,

you can use someone elses!

1 Plasmid
source
tube
Label
exactly
as shown
BMP###

Forward primer: complimentary

to beginning of MCS (template
strand)
Reverse primer: complimentary
to end of MCS (non-template
strand)

forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse
forward
reverse

Primer

2 tubes:
fwd
revs

DO NOT
PUT A
COLORED
CIRCLE ON THE
TUBES

#2: Sanger Sequencing

This video can be seen at https://www.youtube.com/watch?v=e2G5zx-OJIw

Thermofisher/LifeTechnologies
More information from Thermofisher/LifeTechnologies
Sanger sequencing handbook: https://tools.lifetechnologies.com/co...
If you have more questions on sequencing, submit your question at https://www.thermofisher.com/ask

There are lots of helpful videos on Youtube, just dont get distracted

Order of Operations
1. Plasmid Prep of pET15b/LDH
1st partner does plasmid prep
2nd partner sets up NAD+ cocktails, labels all
cuvettes for kinetic assays

2.
3.
4.
5.

Set up RE digestions
Perform LDH kinetic/inhibition assays
Run gel
Make sequencing reactions