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!!