The characterization of Amino Acids and the Purification of

Proteins

Shawndell PowellHigh School for Medical Science

Bronx, NY, 10457

Dr. Jeffrey O. BolesChemistry Department

Tennessee Technological UniversityCookeville, 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:

H2NRC C

H

O

OH

Proteins• A large number of organic compounds that

make up living organisms and are essential to their functioning.

• Whether found in humans or in single-celled bacteria, proteins are composed of units of about 20 different amino acids

To make selenatryptophan more commercially available

Run a TLC (Thin Layer Chromatography)

Purify Proteins

Objective:

Goal:

RxN

Se-Indole analog substratesynthesized

at Los Alamos National Labto make with 90%

yield

the enzyme that were over expressedin bacteria and then purified using

medium pressure liquid chromatography

commercially available buffer to control pH

product analysis

byTLC &NMR

selenatryptophan

centraprep toremove enzyme

betteryield

reproducibility

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

• 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 2-3mL of H20 or 10% EtOH through the column

• Turn P-1 off, switch to Buffer A, turn on P-1 on for 2-3mins.

Wash Mode:

• Drop A & B line of P-50 into Buffer A and Buffer B, change to “wash” mode

• Run method 0 on gradifrac

P-50 Preparation:

• 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):

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 10-30mins.(until the recorder approaches the base line

• Press “end” on gradifrac, turn off P-1• Change valve to “load” mode and run method

• 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

Stripping Chelating Sepharose:

• 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.

After finishing your chromatography:

Acknowledgements• Dr. Boles• Jeffrey• Dr. Dan• Dr. Sat• Harlem Children Society• Chemistry Dept. at TTU