Size Exclusion Chromatography

Proteins

•75% of dry matter in living things is protein.

•Biologist must purify protein from other proteins in the cell.

•What are characteristics of Proteins we could use to separate them?

Characteristics of Proteins

•Hydrophilic•Hydrophobic•Positive•Negative•Size•Active site

Amino Acids

HydrophobicHydrophilic

Size

Active Site

•Biological function

•Individual chemical structure

Chromatography

•Allows the separations of individual component from complex mixtures

•Biology– Separation of chlorophyll

•Biotechnology–Purify biological molecules

Separation of Chlorophyll

Protein Purification

•Affinity Chromatography•Ion Exchange

–Anion exchange–Cation exchange

•Gel Filtration Chromatography–size exclusion chromatography

Affinity Chromatography

•Biological function

•Individual chemical structure

Antibody isotypes of mammals

Name Types DescriptionAntibod

y Complexes

IgA 2

Found in mucosal areas, such as the gut, respiratory tract and urogenital tract, and prevents colonization by pathogens.[10] Also found in saliva, tears, and breast milk.

IgD 1

Functions mainly as an antigen receptor on B cells that have not been exposed to antigens.[11] Its function is less defined than other isotypes.

IgE 1

Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms.[6]

IgG 4

In its four forms, provides the majority of antibody-based immunity against invading pathogens.[6] The only antibody capable of crossing the placenta to give passive immunity to fetus.

IgM 1

Expressed on the surface of B cells and in a secreted form with very high avidity. Eliminates pathogens in the early stages of B cell mediated (humoral) immunity before there is sufficient IgG.[6][11]

1. Affinity Chromatography

•Loading affinity column.

2. Affinity Chromatography

•Proteins sieve through matrix of affinity beads.

3. Affinity Chromatography

• Proteins interact

with affinity ligand with some

binding loosely

and others

tightly.

4. Affinity Chromatography

• Wash off

proteins that do not bind.

5. Affinity Chromatography

• Wash off proteins

that bind

loosely.

6. Affinity Chromatography

• Elute proteins

that bind

tightly to

ligand and

collect purified protein

of interest.

Affinity Chromatography Animation

Animation

Affinity Chromatography

Ion Exchange Chromatography

•separates biomolecules based on differences in their –anionic –cationic

•charge characteristics

Ion Exchange Chromatography

•Anion exchange

•Cation exchange

Gel Filtration Chromatography

Gel Filtration Chromatography Animation

Animation

Hemoglobin•Brown•65,000 Daltons•4 sub units

Hemoglobin

Vitamin B12

•Pink•1350 Daltons

Vitamin B12

1. Obtain 12 collection tubes and label ten sequentially from 1 to 10.

2. Label the final two tubes “Waste” and

“Column Buffer”. 3. Using a clean pipette, transfer 4

ml of column buffer into the tube labeled “Column Buffer”.

Column Bed

Vocabulary

•Buffer–Liquid used to dissolve biomolecules

•Fractions –Samples collected

4. Remove the cap and snap off the end of the sizing column. Allow all of the buffer to drain into the waste tube.

5. Cap the bottom of the column.

6. Place the column onto tube 1.

7. You are now ready to load the protein sample onto the column.

8. Uncap the column. **It is important to uncap the

column only when you are ready to load your protein—you do not want your column to run dry.

9. Using a pipette, add one drop of protein mix onto the top of the column bed.

**The pipette should be inserted into the column and the drop should be loaded just above the top of the column so that it minimally disturbs the column bed.

10.As soon as the drop of protein mix enters the column bed, add 250 μl of column buffer to the top of the column.

11.Let the buffer run down the side of the tube and onto the top of the bed.

12.Begin to collect drops into tube 1.

13.Add another 250 μl of column buffer to the top of the column.

14.Continue to collect drops into tube 1.

15.Add 3 ml of column buffer to the top of the column matrix.

16.Transfer the column to tube 2 and count the drops that enter into each tube.

17.Collect 5 drops of buffer into tube 2.

•Hemogloblin moves faster through the column than the Vitamin B-12

18.When 5 drops have been collected into tube 2, transfer the column onto tube 3.

19.Collect 5 drops of buffer into each tube.

20.When 5 drops have been collected into a tube, lift it off and transfer it to the next tube.

21.Continue collecting 5 drops into each tube.

22.When you reach tube 10, collect a total of 10 drops.

23.Sketch your results.

Results