Proteomics Ion Exchange Chromatography Ion Exchange Chromatography Ion exchange chromatography is a purification technique, which involves the separation of the proteins based on the ion exchange property between the proteins and the column. Learning Objectives: After interacting with this learning object, the learner will be able to: Appreciate buffer preparation. Prepare ion exchange column. Elute and purify the required proteins from the sample. Note: The current IDD exists in two modes- interactive and automatic. Students taking lab course should select interactive (set as default), while the automatic mode may be selected for general users.
Sample Preparation Measuring cylinders are available at different volumes. Based on the user s requirement a specific volume can be measured using these cylinders.
Sample Preparation Place the paper on the electronic balance and tare its weight before weighing.
Sample Preparation Prepare DEAE-sepharose and CM-cellulose resin. DEAE is used to prepare the anion exchange column while CM cellulose for cation exchange column. After weighing required amount of resins based on the column length, the volume of the water to be added to suspend the resin is optimized.
Sample Preparation Prepare 10mM phosphate buffer of ph7.2 using monosodium phosphate and disodium phosphate and water and mix thoroughly.
Sample Preparation The magnetic stirrer is used for evenly distribution of solute into the solvent.
Sample Preparation Prepare 50mM, 250mM and 500mM of High salt buffer solution containing NaCl. The buffers of different strengths are required for various stages of chromatographic procedure.
Resin/Buffer Preparation Before measuring the ph, the ph instrument should be calibrated with standards, first with STD-1 at ph 4 and then with STD-2 at ph 9.
Resin/Buffer Preparation Adjust the ph of the Phosphate buffer to 7.2 using NaOH. NaOH is used to increase the alkalinity of the solution so the ph increases on adding.
Resin/Buffer Preparation Prepare Phosphate buffer of ph 7.2.
Sample Addition Pack the columns by DEAE and CM cellulose resins. Avoid air bubbles, void spaces during the column packing.
Sample Addition Equilibrate the column using Phosphate buffer of ph 7.2, to achieve a constant environment throughout the column.
Sample Addition Take the stored sample and thaw it. Load the sample to both anion exchange and cation exchange columns for separation.
Elution Cationic proteins bind to CM-cellulose resin while anionic protein bind to DEAE resin based on the strength of charge interaction.
Elution Pour the low ionic strength mobile phase through the column.
Elution The elution step helps for the collection of the sample at different fractions.
Elution Collect the eluted sample. Proteins with low ionic interaction will be eluted first and when the ionic strength is increased proteins with high ionic interaction will be eluted out.
UV Spectrometry Detect the presence of protein by using UV-visible spectrometry. The high absorbance reading indicates the presence of protein.
UV Spectrometry Draw the chromatogram graph with absorbance at Y axis and volume of eluent in x axis to determine the protein from the literature or to know the presence of protein. The sample collected can be directly injected into the LC-MS analysis. For further information please go through following IDDs.