Isolation and Characterization of
Cellular Complexes Containing the
Histone Deacetylase SIRT1
Vincent Lew
Howard Hughes Medical Institute Fellowship
Mentor: Dr. Mark Leid
Molecular Pharmacology Lab
College of Pharmacy - Oregon State University
DNA
 Humans have 14,000 to 73,000 micrometers of DNA per
chromosome
 All 46 chromosomes together in a human cell have 2
meters of DNA which is contained in a nucleus that is 5
micrometers in diameter
How does it fit?
How does it fit?
History
 Human Jurkat Cells (Immune System)
 Silent Information Regulator (SIRT)
 Chicken (Ovalbumin Upstream Promoter)
Transcription Factor Interacting Proteins 1
& 2 (CTIP1 & CTIP2)
 Histone DeAcetylase Complex (HDAC)
Represses Gene Transcription
Project Aim
 To isolate Silent Information Regulator
(SIRT) protein & complexes via purification
 To investigate all of the functions of SIRT
in the human body
 Determine by finding what proteins are
found with it
?
?
SIRT
?
Activation
RNA
Polymerase
RNA
Polymerase
Acetylation
Complex
Acetylation
Complex
Acetylation
Complex
Repression
RNA Polymerase
CTIP / SIRT
Complex
CTIP / SIRT
Complex
Step One: Phosphocellulose
 Phosphocellulose
Cl-
IN
Na+
Column
 Tightly packed resin
 Negatively charged
 Wash w/ increasing
NaCl molarity
(stronger washes)
ClNa+
COLLECT
Step Two: Where is the Protein?
 Electrophoresis
 Negatively Charged & Migrates to Cathode
 Smaller strands migrate faster
10% Acrylamide
Gel
-
+
Step Two Continued
 Electrotransfer
 Use of electricity to transfer proteins from
acrylamide gel to nitrocellulose membrane
10% Acrylamide
Gel
Nitrocellulose
Membrane
Step Three: Western Blot
 Immunoreactivity
 Use of antibodies that bind to specific proteins
specific
protein
chemiluminescence
nitrocellulose
membrane
Western Blot Results
Input
Flow
Through
300
mM
600
mM
What does this mean?
• There is a significant amount of SIRT protein in the Input &
Flow Through
• Input: originally put into the column (before purification)
• Flow Through: protein that did not stick to the column
Step Four: DEAE
 Diethylaminoethyl
Cl-
IN
Na+
Column
 Tightly packed resin
 Positively charged
 Wash w/ increasing
NaCl molarity
(stronger washes)
ClNa+
COLLECT
Western Blot Results
Flow
Through
Input
400 mM
200 mM
800 mM
600 mM
1M
What does this mean?
• There is SIRT protein in the Input, .2mM & .4mM lanes
• Input: originally put into the column (before purification)
• .2mM: what eluted after .2 milli-Molar NaCl solution
• .4mM: what eluted after .4 milli-Molar NaCl solution
Step Six: Size Exclusion
 Size Exclusion column
 Separates based on SIZE of molecules
 Use of known markers to determine size of
unknown molecules
 Thyroglobulin: 669 kD
 Catalayse: 232 kD
 BSA (albumin): 67 kD
 1 kiloDalton = 1.67 x 10-24 kg
Size Exclusion
IN
OUT
Western Blot Results
Thy
(669)
Cat
(232)
BSA
(67)
200 mM
Thy
(669)
400 mM
Cat
(232)
Step Seven: Immunoprecipitate
 Final step of purification in this project
 Use of specific antibody (Abx) to bind to
individual protein complex
 Extract only that which is bound to antibody
Immunoprecipitation
Sepharose
Beads
Sir2 Abx
Protein X
Protein Y
SIRT
SIRT complexed to
unknown protein
Mass Spectrometry
 Determine protein structure based on
molecular mass
Results
 Still to be determined
Acknowledgements
 Howard Hughes Medical Institute
 Dr. Mark Leid – Molecular Pharmacology,
Oregon State University
 Valerie Peterson – Molecular Pharmacology,
Oregon State University