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 Hydrophobic Hydrophilic

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 NameTypesDescription Antib ody Com plex es IgA2 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.mucosalgut respiratory tracturogenital tractpathogens [10] IgD1 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. [11] IgE1 Binds to allergens and triggers histamine release from mast cells and basophils, and is involved in allergy. Also protects against parasitic worms. [6]allergenshistaminemast cellsbasophilsallergy parasitic worms [6] IgG4 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. [6] IgM1 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] [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 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 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