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5.36 Biochemistry Laboratory: Mit Opencourseware
5.36 Biochemistry Laboratory: Mit Opencourseware
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5.36 Lecture Summary #6 March 17, 2009
Upcoming CI-M due date: The final draft of your mini-review is due at noon on
Tuesday, March 31st. Reviews should be submitted on the MIT class website.
Please note that there will be a 5-point penalty for late submissions. An additional 5 points
will be deducted for each subsequent day that the assignment is late.
OH N O O- N
N N
-O
O O O
N
N
kinase -
O O
N
N
P O P O P O O P O P O
O O
substrate O- O- O-
H
H H
H substrate
O- O-
H
H H
H
OH H OH H
ATP ADP
Why are scientists interested in detecting kinase activity?
• Enables monitoring of kinase activity and inhibition
• Used for screening of ___________________
• Facilitates the unraveling of complex cell-signalling pathways implicated in
processes ranging from cell cycle regulation to cellular ______________
(including tumor metastasis).
consumption
N
-O 32P O P O P O O O N
O
H H -O P O P O
O- O- O-
O
H H
substrate OH H
substrate O- O-
H
H H
H
OH H
Following ______________ of the unreacted [γ-32P]ATP, the incorporation of 32P into the
substrate is quantified on a scintillation counter.
The requirement for residual ATP separation is the major drawback of this method.
Additional assay considerations (both negative and positive):
• generates ________________ waste
• non-continuous, meaning that it is necessary to take time points if you need
kinetic data.
• General and sensitive. The assay works for ______ kinase.
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Note: 32P incorporation (or other in-vitro) assays can be applied to cell lysate if the
kinase of interest is first purified from the cell lysate by _________________________.
Immunoprecipitation is the use of an _____________ to specifically bind a target
protein and provide a handle for protein isolation.
incubate cell
lysate
wash
substrate + 32P-substrate
32P-ATP + ADP
ADP
Ribo
N
Through two enzymatic reactions, Abl kinase activity is coupled to the conversion of
__________ to ________.
The specific activity of the kinase can be calculated based on the decrease in NADH
absorbance at _______ nm over time. The ε340 of NADH = 6220 cm-1M-1.
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Beer’s Law: Abs = εcl where c = concentration, and l = pathlength of the cuvette
The protein kinase catalyzes the transfer of a phosphate group from ATP to a T, S, or
Y on the peptide substrate. For our Abl assays, the substrate is EAI___AAPFAKKK.
NH3+ NH3+
-O
O-
O O O P O O O
OH H H H O H H H
N N N O
N N N N N
N N NH2 N N NH2
O H H H H
O O O O O O O
Abl kinase
O
HN
NH O
ATP O
HN
NH O
________
NH O +H
3N NH3+ NH O +
H3N NH3+
HN HN
O O
NH O NH O
AcHN
substrate AcHN
P-substrate
O O-
O O-
OH OH
O PK OH
-O P O
O ADP HO
ATP O O
O
O-
PEP pyruvate
You will monitor the ________ of NADH absorption at _____ nm to quantify the
__________________ of wt and H396P Abl kinase domain
These probes comprise a substrate peptide that includes an amino acid recognition
sequence for a specific kinase and an appropriately positioned _________________
sensitive or ______________-enhanced fluorophore.
A) Environment-Sensitive Fluorophores
The signal intensity and maximum emission wavelength of environment-sensitive
fluorophores (also referred to as solvatochromic dyes) are affected by the __________
of the fluorophore’s immediate environment.
SH2 domain
OH OPO32- OPO32-
kinase SH2 domain
F F F
ATP, Mg2+ substrate (pTyr binding domain)
substrate
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B) Chelation-Enhanced Fluorophores
Chelation-Enhanced Fluorophores demonstrate dramatic fluorescence intensity
increases upon metal chelation.
In CHEF-based kinase activity probes, the probes have low affinity for Mg2+ in the
non-phosphorylated form. Once phosphorylated the binding affinity increases
drastically, resulting in a ____ to _____ fold fluorescence enhancement.
OH
O
Me2NO2S N
Me2NO2S N
kinase Mg2+
O
S
OH ATP, Mg2+ S O P
O
O
S/T/Y
S/T/Y
This is an ideal assay for cell lysate applications. Adjustments in the sensors to
optimize detection with lower levels of MgCl2 (initial conditions required 10 mM
MgCl2, above physiological levels) will enable in-vivo applications.
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III. IN-VIVO PROTEIN-BASED PROBES (ie. FRET)
Protein probes for sensing kinase activity typically employ fluorescence resonance
energy transfer (________) to sense a conformational change induced by protein
phosphorylation.
When the donor and acceptor fluorophores are in close proximity (10-100 A), the
acceptor fluorophores is excited by the donor emission and emits a signal*.
Protein engineering has led to the development of a rainbow of other GFP analogs
with a range of excitation and emission wavelengths. All are _______________ , but
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FRET-based phosphorylation sensors: Detection of a phosphorylation-induced
______________________ change
For example, below is a cartoon of a FRET-based sensor for protein tyrosine kinases
(PTK) using the CFP/YFP FRET pair.
The major advantage to FRET-based probes is that they can be expressed in cells.
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IV. LABORATORY REPORT GUIDELINES
Grading Rubric for the Laboratory Report / Lab Work: 50 points total
Abstract: 5 points
Introduction: 10 points
Appendices: 12 points
Remember: You want to impress us with your understanding of the background, the
techniques, and the chemistry involved.
______________________________________________________________________
General Outline of the Module 4 and 5 Laboratory Report:
Please use the following subheadings in your report.
Abstract
This should provide a concise summary of the purpose of the report, major
experimental data obtained, and major conclusions. The length should not exceed
250 words.
Introduction
This serves to introduce the reader to the report that follows. The introduction
should also emphasize the relevance and importance of the experiment.
Your introduction should include:
• Any background information that is required to orient the reader to the
experiment.
Do not use the first person. The introduction should include references from any
pertinent literature if necessary (the first journal reading assignments from Lectrue
#1 should provide sufficient information for your introduction). Please reference as
in Nature.
Experimental Results
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Describe the results. Refer the reader to any specific figures, tables, or appendices.
All essential figures must go in this section. The goal of this section is to simply
report the results. DO NOT discuss or interpret the data here.
Conclusions
This is a BRIEF summary of the project goals and major findings. This should be one
short paragraph, concise and direct.
References
Again, please follow Nature reference formatting. It is ok to have only 1 reference.
3) Supplementary Data
This section is for any non-essential data/figures that aren’t included in the
lab report (such as std curve fits, absorbance readings, sequence information,
etc.)
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