Protein purification (Dr. Miles TAMU)

Question 1

Steps for protein purfication

Answer 1

1. protein source
2. Lysing the cells
3. Assay

Question 2

What are the 4 different ways for lysing cells

Answer 2

• hypotonic solution
• Sonication
• French press
• Lysozyme

Question 3

How do we lyse open animal tissues

Answer 3

By osmotic lysis, by placing cells in hypotonic solution, the osmotic pressure forces water into the cells causing them to swell and rupture

Question 4

Sonication

Answer 4

Ultra high frequencies of sound breaks open cell walls.

Question 5

French Press

Answer 5

Uses high pressure to force cells through a pinhole opening the sheer causes the cells to lyse open.

Question 6

Lysozyme

Answer 6

Enzyme that breaks down bacteria’s cell walls.

Question 7

Two steps in lysing cells

Answer 7

1. is actually lysing the cell
2. Centrifuge down the cell debris

Question 8

What is used to centrifuge done proteins

Answer 8

Serine and cystine proteases degrade proteins so inhibitors are added

Question 9

Salt fraction steps

Answer 9

• Salting in: The solubility f the protein increases as the salt concentration is raised
• Salting out: proteins solubility decreases as the salt concentration increases

Question 10

Column Chromatography

Answer 10

Takes advantage of charge, size, binding affinity, polarity
- Protein containing solution will stay on top
- I twill form bands based of each protein

Question 11

Size exclusion chromatography

Answer 11

-Also called gel filtrations
- Gel will separate proteins based on their size
- Bigger proteins(heavy) will elute faster than smaller proteins (light) according to their weight

Question 12

Ion exchange chromatography

Answer 12

Difference in size and magnitude of the net electric charges of proteins at a given pH
- in cations, the positives will stay at the top and negative charges will fall faster
- in anion, the negatives will stay at the top and the positives will fall faster

Question 13

Affinity Chromatography

Answer 13

Ligand will form to the protein of interest causing it to fall slowly through the chromatography while everything else will fall faster

Question 14

Dialysis

Answer 14

Separates molecules based on size according to the permeable membrane
- This is a way of removing ammonium sulfate after a salt fractions or for changing from one buffer to another

Question 15

Electrophoresis

Answer 15

Determines the number of proteins present and estimate the purity of their proteins
- Allows determination of molecular weight a pI

Question 16

SDS-PAGE

Answer 16

Detergent that denatures proteins into rods
- binds through hydrophobic interactions in ratio of 1 SDS per amino acid
- Separates solely based on weight

Question 17

Proteins will keep migrating till when

Answer 17

where the pH equals the proteins isoelectric point where there will be no net charge