Each column filter or stopcock for the column stopcock to

Last updated: 10/5/2023

Each column filter or stopcock for the column stopcock to your lab bench 1 Equilibrate the columns by applying 30 mL of Buffer A through the column DO NOT let the column dry out always add the next buffer immediately after the previous buffer runs completely into the column 2 While the columns are equilibrating centrifuge any solid precipitate from your dialyzed samples P1 and P2 by spinning in the tabletop bucket centrifuge at 1000 rpm for 3 min you will need to separate the sample into several Eppendorf tubes Transfer the supernatants to fresh Eppendorf tubes do not disturb the solid precipitate at the bottom Set aside 1 mL of P1 and P2 for later use during protein electrophoresis 3 Dilute the remaining P1 sample 100 fold in DI water Load 3 5 mL of each sample diluted P1 and undiluted P2 onto separate columns Allow the samples to flow into the column 4 Wash columns by passing 10 mL Buffer A through the column 5 Load 10 mL of the low salt buffer Buffer A 50 mM NaCl onto the column After 5 mL of salt buffer has flown through entered the column start collecting fractions of 1 2 mL each in cuvettes set a cuvette below the column and let the drops fall in until the cuvette is three fourths full for each salt buffer you will fill approximately 5 cuvettes BE SURE TO NUMBER THE FRACTIONS IN ORDER 6 Blank the scanning spectrophotometer with the low salt buffer As you collect fractions measure the OD 280 nm with the spectrophotometer Proteins absorb UV light at 280 nm so those fractions containing the most protein should have the highest OD 280 nm When you identify the fraction from the low salt wash that contains the most protein first perform a spectral analysis on the sample and then transfer 2 mL of this sample or as much as you can to a labeled Eppendorf tube and place on ice to be used later The remaining samples can be discarded 7 Load 10 mL of medium salt buffer Buffer A 200 mM NaCl onto the column As before collect 1 2 mL fractions into labeled cuvettes and determine which fraction contains the most protein by reading the OD 280 Blank with medium salt buffer Once you have identified the fraction from the medium salt wash that contains the most protein first perform a spectral analysis of the sample and then transfer the sample to a labeled Eppendorf tube and place on ice for later use The remaining samples can be discarded 8 Load 10 mL of high salt buffer Buffer A 500 mM NaCl onto the column Collect 1 2 mL fractions into labeled cuvettes and determine which fraction contains the most protein by reading the OD 280 Blank with the high salt buffer Once you have identified the fraction from the high salt wash that contains the most protein first perform a spectral analysis of the sample and then transfer the sample to a labeled Eppendorf tube and place on ic for later use The remaining samples can be discarded