BL4820 Lecture 6 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3
Part C
6. Changes to Week #6 Lab Procedures
These procedure changes will be handed out in class and also in the Lab (in case you
miss the class) - be sure to put this information in your Lab Report in the Methods
section.
Carboxy Methyl Cellulose (CMC) Column Chromatography
- The dimensions of the CMC column will be different from those given in the text. We will
use a column 2.5 x 1.7 cm. The column will be ready when you arrive in the Lab. It will
have a small layer of glass wool on top the CMC to make it easier for you to layer your
GOT sample and the various elution fractions without disturbing the CMC bed.
- Set up 20 test tubes numbered 1 to 20. All test tubes must be of the same diameter. Also
set up two test tubes: one containing 2.5 ml of water and one containing 5 ml water. These
tubes will be used for comparison to your fractions during the elution where you collect
the column effluent so that you can collect fractions of 2.5 and 5 ml volumes.
- The first step will be to drain off excess buffer from the top of the column by opening
the outlet and letting the buffer flow into a beaker. This buffer is discarded.
- Next apply 1.5 ml of your centrifuged AS-II fraction which has been dialyzed. Be sure to
save some of this sample for analysis of GOT activity and protein content. As you layer
the sample on, be sure to avoid upsetting the CMC bed and get the sample evenly
distributed over the column. While the sample runs into the CMC bed, begin to collect
fraction #1. Let it run in slowly!!! Allow all to run in before you do the next step.
- Apply the 25 ml of 0.03 M Na-Acetate, pH 5.0, wash buffer which will wash the column of
non-binding proteins, to the top of the column very carefully so you do not disturb the
CMC bed. Be sure your sample has completely entered the column before applying this
buffer. Continue to collect fraction #1 until you have 2.5 ml and then collect 2.5 ml
fractions until all the wash buffer has entered the column.
- Apply 50 ml of 0.08 M Na-Acetate, pH 5.0, elution buffer, which will elute the GOT from
the CMC column, to the top of the column and begin to collect fraction #11. This will be a
5 ml fraction and you continue to collect 5 ml fractions until all the elution buffer has
passed through the column.
- Read the A-280 nm of each fraction and record it in relation to fraction #. This will
provide a measure of the protein content of your fractions. The concentration of the
protein in the fractions can be calculated using the following equation: 1.0 A-280 nm =
1.0 mg protein/ml. Thus, if the A-280 nm of a fraction is 0.2 then it has 0.2 mg
protein/ml.
- Assay GOT activity of every other fraction beginning with fraction #2. The most active
fractions will need to be diluted 1:9 or more in some cases, to get the assay to be on
scale. If you get a very active fraction when you have the 1/10 dilution, even if you can
read the rate, try a 1/20 dilution since you may get a better result with this dilution.
In the regions of the elution fractions, do an assay on every fraction so that you find
all those with high GOT activity. So if fraction 12 has GOT activity, then do fraction 11,
12, 13, 14 and 15 for GOT activity.
Save the fraction with the highest GOT activity, which will be analyzed in Weeks #7
and 8 using gel electrophoresis. Give this fraction to your TA with a label with your lab
group name on it.
Back To: Lecture 6 - Week #6
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