Biochemistry 2280A Lecture Notes - Lecture 7: Protein Purification, Enzyme Assay, Recombinant Dna

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22 Oct 2018

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Biochemistry lecture 7 september 24, 2018. Topic 5 protein purification and proteomics: protein purification. Proteins are present in cells or secretions, and are thus complex mixtures usually many different proteins. Need to re(cid:373)o(cid:448)e u(cid:374)(cid:449)a(cid:374)ted (cid:373)aterials (cid:894)(cid:858)(cid:272)o(cid:374)ta(cid:373)i(cid:374)a(cid:374)ts(cid:859)(cid:895) (cid:449)hile preser(cid:448)i(cid:374)g as (cid:373)u(cid:272)h desired protein as possible. Proteins may differ in many properties: size and shape, charge, affinity for different ligands, surface hydrophobicity) Exposes differences in the properties of different proteins to divide them into groups and subgroups: before you begin the purification process. Start with preparative method, follow with analytical: preparati(cid:448)e (cid:373)ethods to ho(cid:373)oge(cid:374)ize (cid:894)(cid:862)(cid:271)ust up(cid:863)(cid:895) (cid:373)aterial. Break open tissue or cells to release protein (undamaged: sonication = high frequency sound waves, grinders of various types, blenders/homogenizers, pressure, detergents. Compatible buffer solution: adjusts the ph so outside influences cannot affect the process and cause damage, o(cid:374)e i(cid:374) (cid:449)hi(cid:272)h the protei(cid:374) is (cid:862)(cid:374)ati(cid:448)e(cid:863, preparative methods centrifugation. Second step is usually centrifugation: spin homogenate to separate by size/density, (cid:862)differe(cid:374)tial (cid:272)e(cid:374)trifugatio(cid:374)(cid:863)

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You purify a protein (a single polypeptide). You produce a monoclonal antibody against your protein and a polyclonal antibody against your protein. You wish to use these two antibodies to determine where in the cell your protein is localized by immune-fluorescence microscopy. Since you've just started working on your protein, you don't know many of its properties, such as whether it acts as a monomer, homodimer, heterodimer, or is part of a larger protein complex. You carry out your experiment and discover that the polyclonal antibody gives a strong signal at or near the plasma membrane and nowhere else. In contrast, your monoclonal antibody gives does not give you any signal. Assuming your protein was not denatured during purification, and that both antibodies are fully functional in your experiment- where do you think your protein localizes, why do your antibodies give you different results, and what do these results suggest about your protein? Assume the results above were for cultured cells that had been incubated for some time in the absence of growth factors (assume this does not trigger apoptosis). You now add a particular growth factor. Two hours later you repeat your immune-fluorescence experiment and find that both the monoclonal antibody and the polyclonal antibody give you signal throughout the cytoplasm. What conclusions and possibilities does this suggest about your protein?

tealcoyote101

This experiment is about Affinity Purification of Green Fluorescent Protein (GFP)

please answer the below questions

1. What is a bacterial colony?

2. Why did you pick one green colony and one white colony from your agar plate(s)? Why do you think you picked one of each color? What could this prove?

3. How are these items helpful in this cloning experiment? a. ultraviolet (UV) light - b. incubator - c. shaking incubator

4. Explain how placing cloned cells in nutrient broth to multiply relates to your overall goal of purifying the fluorescent protein.

5. You have used a bacterium to propagate a gene that produces a green fluorescent protein. Identify the function of these items you need in this procedure. a. Centrifuge â b. Lysozyme â c. Freezer â

6. Can you explain why both liquid cultures fluoresce green?

7. Why did you discard the supernatant in this part of the protein purification procedure?

8. Can you explain why the bacterial cellsâ outer membrane ruptures when the cells are frozen. What happens to an unopened soft drink when it freezes?

9. What was the purpose of rupturing or lysing the bacteria?

10. Briefly describe hydrophobic interaction chromatography and identify its purpose in this lab.

This lab is about Crystal Growth of Hen Egg White Lysozyme so please answer the following question

1. What condition yielded the "best" crystals of lysozyme?

2. What changes did your group try to make, and what effect did they have on the crystals?

3. How many of your conditions yielded crystals? How many did the entire class get? Give the percentage (number of conditions that gave crystals/number of different conditions that were set up) for your attempts as well as for the entire class. What does this tell you about protein crystallization?

4. What methods can you think of to determine if the crystals are lysozyme and not salt (many salt crystals are colorless as well)? (Hint: think of protein properties and ways to assay and look at proteins).

5. Many protein crystals are formed at or near the pI of the protein being studied. Can you rationalize any reason why this observation might be made (in other words, why is the pI so good at helping form protein crystals).

Biochemistry 2280A Lecture Notes - Lecture 7: Protein Purification, Enzyme Assay, Recombinant Dna

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