Lab # 10 Purification of proteins

Isolation separation purification and identification is a very common and important process in Biochemistry. In this lab we will therefore explore how this is done.

After reading the PowerPoint presentation and the protein purification word file provided you can relax and enjoy the videos that I have linked below that describe several of the techniques used in protein purification.

Finally, answer and submit the questions below.

1. How would you define “purity” with respect to purification of proteins?

2. Define purification level

3. Give the mathematical equation that defines purity.

4. List five stages into which protein purification can be divided

5. Explain why scientists purify proteins?

6. You are asked to purify a hexokinase. Discuss the parameters you would consider in sourcing and selecting such an enzyme, and briefly indicate how you would stabilize these proteins on isolation. In answering this question, students should ask and answer the following questions.

A What is a hexokinase?

B Which hexokinase would you think of purifying? Why? Think where would you find lots of this enzyme in a mammal? (You should be able to get this organ easily at the butcher’s from a pig or cow.)

C will you find this enzyme in highest concentration in liver or muscle? Why?

D Now follow the techniques given in the PowerPoint and give details of how you will extract, isolate and purify the enzyme.

E How would you prove that you have indeed extracted a/this hexokinase?

Please note that QUESTION 6 IS WORTH 50% OF THE MARKS FOR THIS ASSIGNMENT

Protein Sequencing –Parts1-4

http://www.youtube.com/watch?v=on7_eVLe5a4&feature=related

http://www.youtube.com/watch?v=u8DXSYwpTpk&feature=related

http://www.youtube.com/watch?v=2MEljBS1SIg&feature=related

http://www.youtube.com/watch?v=pouL7-QEYho&feature=related

ANALYTIC ULTRA CENTRIFUGATION

http://www.youtube.com/watch?v=7ehsuhgtdNU

http://www.youtube.com/watch?v=ZOdSeowX61A&feature=related

http://www.youtube.com/watch?v=Yky4m9_7N9k&feature=related

ELECTROPHORESIS

Gel electrophoresis

http://www.youtube.com/watch?v=o-wh_-tLI70&feature=related

Gel Electrophoresis

http://www.youtube.com/watch?v=QEG8dz7cbnY

http://www.youtube.com/watch?v=68r6FzKYpW8&feature=related

http://www.youtube.com/watch?v=BDadjDRl-Vg&feature=related

http://www.youtube.com/watch?v=K2ni7mDGwAk&feature=fvw

Gel Electrophoresis and Blotting 1&2

http://www.youtube.com/watch?v=K2ni7mDGwAk&feature=fvw

http://www.youtube.com/watch?v=Inc_9lXVNIM&feature=related

SDS-PAGE (polyacrylamide gel electrophoresis)

http://www.youtube.com/watch?v=IWZN_G_pC8U&feature=related

http://www.youtube.com/watch?v=lM8npqHw-mk&feature=related

SDS-Page

http://www.youtube.com/watch?v=0Zf1eggq3Nw&feature=related

http://www.youtube.com/watch?v=NWKbe8Wto6A&feature=related

http://www.youtube.com/watch?v=snJMzL6KG_M&feature=related

http://www.youtube.com/watch?v=EDi_n_0NiF4&feature=fvw

http://www.youtube.com/watch?v=XUjLO-ek2C8&feature=related

http://www.youtube.com/watch?v=YDA57njjdg4&feature=related

SDS PAGE protein separation part 1 & 2

http://www.youtube.com/watch?v=-iKgynQvLDc&feature=related

http://www.youtube.com/watch?v=EckO4R3Z770&feature=related

Western blotting

Western Blot 1&2

http://www.youtube.com/watch?v=BQOOsLDuZaU&feature=related

http://www.youtube.com/watch?v=yZKTJS7L7VQ&feature=related

http://www.youtube.com/watch?v=iUrK9FuMAgs&feature=related

http://www.youtube.com/watch?v=mjbr3beDslo&feature=relatedhttp://www.youtube.com/watch?v=mjbr3beDslo&feature=related

http://www.youtube.com/watch?v=HddVrI2YdZY&feature=related

http://www.youtube.com/watch?v=aGu-NKvKIoA&feature=related

http://www.youtube.com/watch?v=SahgDkDSutk&feature=relatedhttp://www.youtube.com/watch?v=SahgDkDSutk&feature=related

http://www.youtube.com/watch?v=deOvTCXIi1A&feature=related

Protein Identification by MALDI-TOF

http://www.youtube.com/watch?v=iwrPkgGxAH0&feature=related

http://www.youtube.com/watch?v=xh8GGzsc2r4&feature=related

The Mass Spectrometer

http://www.youtube.com/watch?v=VF1nkO1hmVM&feature=related

http://www.youtube.com/watch?v=J-wao0O0_qM&feature=related

http://www.youtube.com/watch?v=OKxRx0ctrl0&feature=related

Isoelectric focusing

http://www.youtube.com/watch?v=0KlSX67onK4

X-ray crystallography

http://www.youtube.com/watch?v=RkRYe1J7cHE

http://www.youtube-nocookie.com/watch?v=YEi54Ir_IJ8