BL4820 Lecture 5 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3
Part B
4. GOT Purification Steps done in Week #5
We will not do the pilot ammonium sulfate precipitation
procedure described in the text since it takes to long to get done within the 3 hour lab
period. We previously determined that the optimum ammonium sulfate precipitation procedure
for concentrating GOT is to prepare the fraction between 50 and 70% saturation of ammonium
sulfate.
So here is what you will do in the lab:
- Put a known volume of your GOT F-II in a beaker on ice and slowly add solid ammonium
sulfate to bring it to 50% saturation while stirring gently. You figure out how much
ammonium sulfate to add using the chart in Table 10-1 in your text on p. 114.
- Stir for 15 min to allow the precipitation process to come to completion and then
centrifuge the solution to collect the protein precipitate. This protein precipitate is
dissolved in buffer and assayed for GOT activity (and protein next week). This solution is
called ammonium sulfate fraction #1 or AS-I.
- Measure volume of the supernatant of the 50% ammonium sulfate precipitation and put it
in the beaker in the ice on the stirrer. Now slowly add enough solid ammonium sulfate to
bring the solution to 70% saturation (again use the chart in Table 10-1 on p. 114 in the
text and be sure you use it correctly) while stirring gently.
- After 15 min of stirring to allow the precipitation process to come to completion,
centrifuge the solution. After centrifugation, the precipitate is dissolved in a minimum
volume of buffer (try to keep it to about 2 ml) and this will be called ammonium sulfate
fraction #2 or AS-II. The supernatant is called AS-III. Both of these should be assayed
for GOT activity and samples saved for protein assays next week.
- Finally, put the AS-II solution in a dialysis bag which has been prepared for you and
clamp it carefully so that it will not leak during dialysis. BE SURE TO PUT A LABEL ON
YOUR AS-II SOLUTION IN THE DIALYSIS BAG SO THAT IT CAN BE IDENTIFIED AFTER DIALYSIS. The
TA will carry out the dialysis over the next two days so that the GOT ASII fraction will
be ready to go for next week's ion-exchange chromatography purification step.
- During the ammonium sulfate precipitation procedure, some members of your group should
carry out Folin protein assays on your F-I and F-II samples from the first week. Be sure
that the samples are sufficiently diluted so that the protein determinations fit on your
standard curve which must be done along with the unknown samples. If they do not fit on
the standard curve, then you can re-do them next week.
Back To: Lecture 5 - Week #5
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