Peptide Purification by RP-HPLC

**Note: This protocol is written with the instrument settings already saved.  Anginex was the designed peptide of interest used for this protocol.**

Peptide Sample Prep and RP-HPLC

Protocol (12/08/11)

By: Sterling Mikkelson

Instrument Used:  Beckman Coulter System Gold 166 Detector

Peptide Sample Preparation:  *note: concentration may vary depending on peptide being purified*

·         Remove peptide powder from -20°C Freezer

·         Weigh out 150mg and add to 50 mL Distilled Water for a final concentration of 3mg/mL

·         Sonicate w/ heat for 15 minutes followed by vortexing until powder is completely dissolved

·         Label 50 mL conical tube peptide solution “Crude Anginex – 3mg/mL” and store at 4°C

HPLC Startup:

·         Be sure solvent lines are placed in appropriately labeled bottles

o   (A = Water + 0.1% TFA;  B = Acetonitrile + 0.1% TFA)

·         Double click 32 Karat Software icon on desktop

·         Double click UV icon

·         Turn on UV lamp by clicking on lamp image (right center of screen)

·         Lamp will switch from blinking yellow to pink when lamp is warmed up

·         Open HPLC startup (File > Methods > Open > startup.met)

·         Click on blue arrow located on the top right

·         In box next to Sample ID, type in t

·         Data Path: D:\public

·         In box next to Data File, type in t

·         Click Start (run will take approx. 19 min.)

HPLC Peptide Sample Run:

·         Open HPLC run (File > Methods > Open > peptide25b.met)

·         Click on blue arrow located on the top right

·         In box next to Sample ID, type in peptidename_date_number of run

·         Data Path: D:\Data

·         In box next to Data File, type in peptidename_date_number of run

·         Click Start

·         Change Flow Rate to 6.00mg/mL by clicking on Flow Rate box (will vary depending on peptide). Click OK

·         Rotate injection knob counterclockwise to the load position.

·         Load syringe with 4.8 mL (max) peptide solution

·         When box in lower right reads “waiting for trigger,” inject peptide solution in injector port

·         With syringe still in the injector port, rotate knob clockwise until it stops then remove syringe

·         Collect samples at each peak over the duration of the run for mass-spec. analysis

·         Print a copy of the run (right click on graph > utilities > print) and label peaks where samples were taken

HPLC Shutdown:

·         Switch solvent lines to bottles labeled 70% Acetonitrile

·         Open HPLC shutdown (File > Methods > Open > shutdown.met)

·         Click on blue arrow located on the top right

·         In box next to Sample ID, type in t

·         Data Path: D:\public

·         In box next to Data File, type in t

·         Click Start (run will take approx. 19 min.)

·         Lamp will automatically shut off

·         Close program by clicking on “X” located at the top right corner of screen