BL483 Biochemistry Techniques

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483 Lecture 4 - Glutamate Oxaloacetic Acid Transaminase (GOT) Purification -- Expt 3 Part A

5. GOT Purification Steps Done in Week #4

Pig Heart extract will be prepared by the TA at the beginning of the lab session this week. You will use this extract to prepare a heated extract with part of the crude pig heart extract and with a small part of it you will prepare a centrifuged crude extract. With the unheated centrifuged crude pig heart extract (called Fraction I or F-I) you will determine how much GOT activity was present before you carried out the heating step. After heating the extract and centrifuging (called Fraction II or F-II), you will determine how much GOT activity remains and so you can figure out how much activity you lost during the heating step. Be sure to save samples of F-I and F-II for protein assays you will do in the next lab. The bulk of F-II is saved for processing in the lab next week.

Be careful during the heating of the extract since the water bath will be near boiling. Also be careful not to over heat your extract since this is a key step in retaining enzyme activity.

Normally, you would not heat an enzyme to purify it because heat denatures most enzymes. But by adding the substrate 2-KG during heating, the GOT is protected from denaturation. Other proteins in the pig heart extract are denatured and precipitate so that GOT is purified by the heating step.

Back To: Lecture 4 - GOT Week #4

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