BISC411
                                          EXPERIMENTAL MOLECULAR BIOLOGY OF THE CELL
 
 

Ion-Exchange Chromatography

Carboxy-methyl sepharose for positively charged proteins and DEAE sepharose for negatively charged proteins.

 Prepare ion-exchange columns. All groups will prepare at least 2 carboxy methyl sepharose columns to purify lysozymes and half will be asked to prepare a third such column for the second protein to be purified. The other half of the groups will prepare one DEAE sepharose column to purify their second protein. Both types of columns are prepared and used in the same way, described below.

1.  Add the appropriate ion-exchange resin slurry to the plastic columns you are given.
    Allow the liquid to drain out. Continue to add more slurry and drain until you achieve a 0.5 ml height of resin in the
    column. Equilibrate the columns by running 3 ml of 50mM Tris, 1mM EDTA, pH 7.3. (equilibration buffer) through
    the column.

2. When your columns are ready, get your dialysate samples from the cold room.

3. Put all of the dialysate onto one of the columns as appropriate (lysozyme onto one carboxy-methyl sepharose column; your other protein dialysate onto the column assigned to your group, either carboxy-methyl or DEAE-dextran. Collect the liquid that flows through into a 15 ml test tube marked as flowthrough lysozyme or flowthrough other protein as appropriate.

4.  Onto another carboxy-methyl column put 500 microliters of a 10 mg/ml lysozyme stock solution (I will provide this). Collect the flowthrough into a microfuge tube labeled control flowthrough. Keep the remaining stock lysozyme for use in
    testing the microccus luteus substrate you will use today (see below).

5. Wash the columns with 3 ml of the equilibration buffer each. Collect 1 ml fractions of eluate (about 10 drops per
    fraction) into microfuge tubes. Put immediately on ice.

6. Then add to the columns 3 ml of 0.05M NaCl in 50mM Tris, 1mM EDTA, pH 7.3..

7. Collect 1 ml fractions from the columns (approximately 10 drops) again. Onto ice.

8. Repeat steps 6 and 7 using 3 ml of 0.3M NaCl in 50 mM Tris, 1mM EDTA, pH 7.3 and with 0.5M NaCl in 50 mM Tris, 1mMEDTA, pH7.3.

9. Remove 200 microliters of each collected fraction from the lysozyme dialysate and lysozyme control columns for preparation of gel samples and protein concentration determination next week. Keep the entire 1 ml of the fractions from the third column. Store all at -20oC.

10. Perform a lysozyme assay using the stock lyzozyme you put aside in step 4. Use the same procedure that you used in week 6. You should get a good drop off per minute. When you know your substrate is working well, test all of the fractions from the two lysozyme columns, control and dialysate. Use 0.5 ml of each fraction in a lysozyme assay with 3 ml of substrate. Your group can divide the work and begin the lysozyme assays while the fractions are being collected. Just remember to save the 200 microliters for next week (see step 9). Record your lysozyme assay data. Can you see any activity in any of the elutions?

Obviously, there is a lot to do today. Plan your work and split up the labor equitably among your group members. The micrococcus luteus substrate will need to be tested (see earlier experiment testing standards) with some of the positive control sample you used for your control column. As fractions come off the columns, you will need to have someone assaying them for lysozyme activity while others continue eluting the protein from the columns. Also, the third column needs to be eluted as well. Be organized!! It is easy to get your tubes mixed up!!