Bio 201: Tutorial

Front 1

protein purification USUALLY involves disruption of the cell followed by column chromatography.

Back 1

chromatography:
a wide variety of technique in which dissolved mlcs is separated thru some type of porous mixture.

Hint 1

AFFINITY CHROMATOGRAPHY

- proteins (such as enzymes) bind specifically to other mlc. Molecules that proteins specifically binds to is called a ligand.

- Protein-ligand binding can be employed by purifying proteins using method called affinity chromatography.

Front 2

chromatography consist of 2 phases:

a) mobile phase - moving solven
b) immobile phase - solvent moving

Back 2

1. mlcs separated are proteins.
2. solvent is a buffered solution
3. immobile phase consist of bead like particle housed in a hollow tube made of glass/plastic.

Hint 2

AFFINITY CHROMATOGRAPHY

- ligand is covalently binded/immobilized to an inert insoluble chromatography bead packed in a glass column.

- mixture of different proteins move down a column containing the immobilized inert insoluble ligand.

- Protein of interest will bind to the ligand whereas all other unwanted proteins pass through the column.

- Free-soluble ligand are then added to column where they complete with the immobilized ligand for binding to protein OR by altering properties of the buffer that influences protein ligand binding.

- bound proteins is then released from ligand by added free ligand that manage to take over the immobilized protein.

Front 3

simplest chromatography:
- aqueous protein mixture is applied to the surface of chromatography matrix at the top of the column.
- mixture moves thru matrix via gravity
- solvent flow is established to carry mixture thru the matrix from top of column to bottom

Back 3

since different mlcs posses different properties, they interact with the matrix differently in terms of rate of flow and are thus separated.

Hint 3

Gel electrophoresis:
- analyze protein fraction and mixtures of proteins

- evaluate quality of purification procedures.

- can do limited characterization of proteins such as estimation of molecular mass.

Front 4

solvent completing the process are collected in tubes and are said to contain fractions of the total sample

Back 4

Protein fragments doe not reflect light and therefore, appear to be transparent and clear.

Hint 4

there are 2 types of gel electrophoresis:
(a) non-denaturing gel electrophoresis (-SDS + beta-mercaptoethanol)
(b) denaturing gel (+SDS + beta-mercaptoethanol) electrophoresis

Front 5

Proteins fragments can be detected because AROMATIC residues can absorb a UV light wavelength of 280nm

Back 5

chromatography --> spectophotometers.

Hint 5

(a) Non-denatureing gel electrophoresis

(-SDS + beta-mercaptoethanol)

- sample electrophoresed through gelatinous material

{method}
1. Gel is first prepared as (l) which is poured into a hollow glass tube (tube gel) or between 2 glass plates (slab gel) and allowed to solidify into gel state. Materials for gel can come from:
(1) single components such as agar or starch gels formed by cooling an initially hot aqueous solution at room temp.

(2) other gels such as PolyAcrylamide gas form thru slow reactions between 2 components initially mixed together and allowed to set.

2. gel formed and is suspended between 2 chambers containing buffer soln.

3. sample containing SEPARATED proteins is added to top chamber.

4. Add sucrose or glycerol in order to increase density of solution and therefore causing sample to sink.

5. Electric current is established to move molecule down the gel separating each other over time due to different migrating rates towards positively charged anode (bottom)

6. Bromophenol blue dye is added to sample to follow the progress of the electrophoresis. Dyes will migrate faster as they are smaller than any protein in mixture.

7. When dye reaches the bottom, it signals the end of the electrophoresis.

8. Slab gels are removed from glass plates and stained to reveal polypeptide (denaturing) or protein bands (non-denaturing)

Front 6

collection tubes are placed in spectophotometers and those that exhibit 280 wavelength absorbance (A280) are retained for further study.

Back 6

Spectophotometer is a tool that measures the amount of light prevented from reaching the photodetector.

The greater the concentration of protein in a solution, the less light that reaches the photodetector, the higher the absorbance reading.

Hint 6

a) non-denaturing gel electrophoresis

factors affecting electrophoretic migration
1. size & mass
-the larger the protein the slower it moves. Molecular level in gel is made up of elastic porous in whihc the proteins must squeeze through.
- Since globular mlc has similar density and shape, size and volume can be equate to mass.

2. charge
- the higher the net charge, the faster the protein moves.

Front 7

4 types of protein purification:
1. column chromatography
2. size exclusion chromatography
3. Ion exchanged chromatography
4. Affinity chromatography

Back 7

SIZE EXCLUSION CHROMATOGRAPHY

- separates proteins depending on the molecular size and volumn

- since most globular proteins have the same density and rougly spherical shape, they are separated on the basis on their MW.

Hint 7

2 ways to separate using non-denaturing electrophoresis gel
- by size, mass and charge
- by pHi (isoelectric focussing)

Front 8

SIZE EXCLUSION CHROMATOGRAPHY

- matrix is composed of porous synthetic beads made of CROSS-LINKED POLYSACCHRIDES.

- Beads of different size can be purchased. The pore size is based on the expected size of the proteins to be separated.

Back 8

SIZE EXCLUSION CHROMATOGRAPHY

- induced volume: the space within the bead
- void volume: the space between the beads.
- void volume is less than the induced volume.
- some proteins are so large that they cannot go through the beads and are forced to go around the beads. Hence, these proteins are guaranteed to leave or "eluted" from the column first.
- The smaller the protein, the more likely it will go thru the beads and stay there longer.
- Due to the porous beads made of cross-linked polypeptides, the size exclusion chromatography elutes proteins in decreasing size order.

Hint 8

Isoelectric focussing (IEF)
- gel electrophoresis which separates proteins on the basis of their pHi.

1. uses polyacrylamide gel to create a stable pH gradient. pH is normally alkaline at the top and acidic at the bottom. Gel is formed using low concentration of acrylamide resulting large pores which do not impede migration of protein

2. as protein moves down the gel, its net charge is constantly changing as a function of the pH.

3. at some point, protein reaches a pH that equals to its pHi. this causes it to stop moving because it possesses a net charge of zero.

- if protein diffuses away from its isoelectric position, it acquires a charge. As a result, it will migrate back to its isoelectric position. This brings to the conclusion that each protein is focused into a narrow region of the gel where the pH= pHi

Front 9

ION-EXCHANGED CHROMATOGRAPHY

- separation is based on average net charge of proteins being separated.

- the higher the avg net charge of the mlc, the stronger its binding to the column.

- when salt concentration is used to displace mlc, proteins are eluted from the ICE in the order of the average charged at the prevailing pH.

- magnitude of the mlc avg net charge is determined by the difference between the protein's pH and pHi.

- The pHi is a useful perdictor of its behavior during IEC.

- IEC depends on the ionic association of proteins with charged groups of the chromatographic matrix.

Back 9

ION-EXCHANGED CHROMATOGRAPHY

- - matrix is composed of (+) or (-) charged beads.

- 2 types of beads commonly used:
(a) diethylamino (DEAE) cellulose : an "anionic exchanger" - it is a positively charged at neutral pH that binds to NET negative charged mlc.

(b) carboxy (CM) cellulose : a "cationic exchanger" - a negatively charged bead that binds to positively net charged mlcs.

Hint 9

Detecting proteins after electrophoresis.

- by staining.
- most common stain: coomassie brilliant blue. - it binds to positively charged arginine and lysine in proteins.

when gel is soaked in coomassie brilliant blue, proteins becomes visible as blue bands in gel. bands indicate non-denaturing

Front 10

ION-EXCHANGED CHROMATOGRAPHY

- when proteins bind to matrix, they are displaced by changing the pH of beads / salt concentration of beads / both

- Displacement of bound proteins can be carried out in 3 ways:
(a) step-wise manner in which a series of solution is added
(b) continuous manner by solutions of continually changing pH OR salt concentration using a TWO CHAMBER GRADIENT MAKER that mixes 2 solution of different composition.

- Changing the pH alters the avg net charge on the mlc affecting its binding to the bead. If the pH = pHi of the protein, the protein is displaced from the matrix because it carrieds no net charged.

- Changing the salt concentration adds ions which compete with the proteins for sites on th matrix. This competition displaces the protein from the matrix.
+++ the higher the avg net charge of the mlc, the stronger its binding to the matrix, the higher the salt concentration required to displaced it.
+++ when salt concentration is used to displace mlc, proteins generally eluted from the ICE in the order of the average charged at the prevailing pH.

Back 10

ION-EXCHANGED CHROMATOGRAPHY

- protein possessing the same net charged as the matrix will not bind to the matrix.
- it passes thru the column "leaving behind" proteins that do bind to the matrix.

- Purification can be facilitated if a pH and matrix can be chosen such that proein of interest do not bind to the matrix while most of the others do.

Hint 10

(b) denaturing gel electrophoresis

same apparatus and similar procedure

- difference is that before the denaturing gel electrophosis, protein is treated with urea, sds or beta mercaptoethanol or combination. they are incorporated in gel.

diff #2 - reveals individual unfolded polypeptides (denatured proteins) and not proteins. "stained bands"

Front 11

SDS-PAGE
* estimate the molecular mass of the polypeptide
* migrate down the gel only via their molecular mass.
*@100C eliminates any shapes differencces between polypeptides. SDS will cause the poly

Back 11

yup.