Download The characterization of Amino Acids and the Purification of Proteins

The characterization of Amino
Acids and the Purification of
Proteins
Shawndell Powell
High School for Medical Science
Bronx, NY, 10457
Dr. Jeffrey O. Boles
Chemistry Department
Tennessee Technological University
Cookeville, TN, 38505
Amino Acids
• A class of organic compounds that contain both
the amino and carboxyl groups.
• Of these acids, 20 serve as the building blocks
of proteins .
• Known as the standard, or alpha, amino acids,
they comprise serine and tryptophan which are 2
of the 20 that are constructed according to a
general formula:
H2N
R
O
C C
OH
H
Proteins
• A large number of organic compounds that
make up living organisms and are
essential to their functioning.
• Whether found in humans or in singlecelled bacteria, proteins are composed of
units of about 20 different amino acids
Goal:
To make selenatryptophan more
commercially available
Objective:
Run a TLC (Thin Layer Chromatography)
Purify Proteins
the enzyme that were over expressed
in bacteria and then purified using
medium pressure liquid chromatography
commercially available
buffer to control pH
Se-Indole analog substrate
synthesized
at Los Alamos National Lab
to make with 90%
yield
better
yield
reproducibility
RxN
product
analysis
by
TLC &
NMR
centraprep to
remove enzyme
selenatryptophan
Running a TLC
• Dissolve 10mg (0.01g) into 2mL
of dH2O
• Vortex until in solution
Running a TLC
• Spot sample on a TLC plate
• Set in TLC solution for about 10-15mins.
Running a TLC
• Look at it under a light
• Circle samples movement
Running a TLC
• Set it in a jar of iodine or spray it with
ninhydrant
• Then dry it with a plow dryer
Results
Purifying Proteins
Wash Mode:
• Turn on recorder, detector, gradifrac, and P-50
pump. Put on recorder pens
• In “wash” mode with the P-1 pump, pass H2O of
10% EtOH through P-1 out port 4 to waste, then
turn P-1 off
• Change to “inject” mode, turn P-1 on and pass 23mL of H20 or 10% EtOH through the column
• Turn P-1 off, switch to Buffer A, turn on P-1 on for
2-3mins.
P-50 Preparation:
• Drop A & B line of P-50 into Buffer A and
Buffer B, change to “wash” mode
• Run method 0 on gradifrac
Loading CFE (Cell Free Extract):
•
•
•
•
Change to “inject” mode
Put P-1 line in Buffer A and pump 2mls
Turn off P-1 pump
Go into manual mode on gradifrac and set
it to 0% B, 2ml/min, 8ml fractions, and put
line 6 in Buffer A after line 6 spends 2mins
in waste beaker
Loading CFE (Cell Free Extract):
• Pump Buffer A through P-1 to equilibrate the
column. Turn P-1 off and press “pause” on
gradifrac
• Put P-1 line in CFE, turn on P-1, press
“continue” on gradifrac and load the CFE, turn
off P-1 and switch line back to buffer A
• Continue with Buffer A through P-1 for 1030mins.(until the recorder approaches the base
line
• Press “end” on gradifrac, turn off P-1
• Change valve to “load” mode and run method
Stripping Chelating Sepharose:
• Push “end” on gradifrac
• Set valve to “inject” mode
• Pump P-1 line in strip buffer and turn on
P-1 on
• Pump until column is white
• Turn off P-1 and change line to 10% EtOH
• Run 10% EtOH through column for 10mins
After finishing your chromatography:
• Push “end” on gradifrac
• Change valve to “inject” mode, put P-1 line
in 10% EtOH and turn P-1 on for 10mins.
Turn P-1 pump
• Change valve to “wash” mode, drop P-50
A & B lines into 10% EtOH and run
method 9 on gradifrac
• Turn all power off.
Acknowledgements
•
•
•
•
•
•
Dr. Boles
Jeffrey
Dr. Dan
Dr. Sat
Harlem Children Society
Chemistry Dept. at TTU