Mahdi Sharawi

Amani nofal

27

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Dr. M. Ahram

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Protein Analysis

· Ion-Exchange Chromatography:

- This method allows for the separation of proteins based on their

charge.

Further Explanation: Proteins have different isoelectric points, so at certain pH

some proteins are positive and some are negative, depending on this information

we determine what kind of bead we need to use to filter out that protein either a

positive or negative bead.

1. Cationic-exchange chromatography: - A Method which involves usage of negatively charged beads. - Positively charged proteins will bind to these negatively charged beads, but

negatively charged proteins won't bind to these beads causing them to be

washed away. - Positively charged proteins are now bound to the negative beads, so we begin

increasing the pH Or NaCl concentration, causing these positively charged

proteins to lose their positive charge which causes them to be Eluted.

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2. Anionic-Exchange chromatography: - A Method with similar concept to cationic-exchange chromatography,

but it uses positively charged beads. Ex: Diethylaminoethyl-cellulose column

- Negatively charged proteins will bind to these positively charged

proteins, but the positively charged proteins won't bind so they will be

washed away. - Negatively charged proteins are now bound to the positive beads, so we

begin decreasing the pH, causing the negatively charged proteins to lose

their negative charge which causes them to be Eluted

Elution:

- The elution method of the past 2 techniques (Cationic and Anionic

Chromatography) is done by the addition of Sodium Chloride ( NaCl )

because sodium ions compete with the positively charged proteins, and

the Chloride ions compete with the negatively charged proteins, for

binding on the column.

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Problem:

You have 5 different proteins (#1, #2, #3, #4, and #5), with different isoelectric points (pIs). pI#5 = 2.3

pI#4 = 4.7

pI#1 = 7.2

pI#2 = 9.1

pI#3 = 12.1

Starting the column at pH 6.5, the sample is added and then washed to remove unbound molecules. What is the order of protein elution in:

A. Cationic-exchange chromatography?

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Answer : 5 à 4 à 1 à 2 à 3

B. An anionic exchange chromatography? Answer: 3 à 2 à 1 à 4 à 5

Answer Explanation:

A. Because we are using Cationic-exchange chromatography, positively charged

proteins will bind to the negatively charged beads, and the negatively charged

proteins will be washed away, Protein 5 is the most negative at pH 6.5 then

followed by Protein 4, so they will be washed immediately, Protein 1 is almost

neutral but has a small positive charge, also protein 2 and 3 are positively charged

but protein 3 is the most Positively charged, So 5 and 4 will be washed away first,

then we begin the elution by increasing the pH causing positively charged

proteins to lose charges and elute one by one.

B. The same concept here but we wash away the positively charged and elute the

negatively charged by decreasing pH.

What's the difference between elution and washing away?

- Elution: Releasing of bound proteins.

- Washing: Releasing of Non-bound proteins.

Affinity chromatography:

- Proteins can interact with other molecules depending on 2 properties,

Affinity and specificity. - Affinity is the strength of binding. - Specificity as in specific for certain molecules. - Affinity chromatography is the separation of proteins depending on

their affinity for a ligand, we use a bead filled column where the beads

are conjugated with these ligands, and only the desired proteins will

bind to these ligands. - Example: The plant protein concanavalin A, which binds to glucose with

high affinity, can be purified by passing a protein mixture through a

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column of beads attached to glucose residues. Concanavalin A, but not

other proteins, binds to the beads. The bound concanavalin A can then

be released by adding a concentrated solution of Free glucose.

Gel Electrophoresis: - Phoresis means movement; Electrophoresis means movement in an

electrical field. - Gel is a semi-solid material which has pores. - This method allows us to separate proteins while they're moving

through the gel in an electrical field. - The electrophoresis technique we're going to use is SDS-Page, (PAGE

stands for polyacrylamide gel electrophoresis)

- How does it work: We put a sample filled with several proteins on top of the gel, and the protein moves through the gel induced by an electrical field, it moves according to size, This gel is a network, the protein will move through this network depending on their size, the bigger the size the slower they will move.

- The gel is made of a material known as polyacrylamide, which is formed

by the polymerization of acrylamide and cross-linked by methylenebisacrylamide

- The gel is made of an Anode(+) and a cathode(-), and the proteins move

from the Cathode to the Anode(- à + ) in the gel, but how does a

positively charged protein move towards the Anode? The presence of

the SDS(Sodium dodecyl sulfate) material, it's made of a hydrophobic

tail and a negatively charged head, this material is a Detergent, it causes

protein denaturation, so the hydrophobic tail enters the hydrophobic

region of the protein, and it nullifies the proteins charge and it becomes

negatively charged because of SDS head.

- Since SDS caused the deformation of the proteins in the gel, we lost the

shape and charge criteria and the proteins will move according to their

Size.

- Native gel: doesn’t include denaturation of the protein and they move

according to size and shape &charge.

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- Proteins which are made of several polypeptides (Dimers, Trimers…etc)

will break up if they're bound by non-covalent interactions, however if

they're bound by covalent interactions like disulfide bonds, we need to

assistance of a reducing agent to break it down.

- When an electrical voltage is applied between the upper and lower ends of the gel, all proteins move in one direction towards the anode (positive) according to size only

After going through all the protein analysis techniques, this is what the final results look like:

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Questions ( refer to slides for pictures )

Describe the protein’s structure based on the following results of SDS-PAGE: 1.Under non-reducing condition, a protein exists as one 40-KDa band. Under reducing conditions, the protein exists as two 20-KDa bands.

Answer: This protein is a dimer, consisting of 2 polypeptide chains; they're bound covalently via disulfide bonds, in the non-reducing SDS-page electrophoresis, they remain intact and create 1 band of 40 KDA, in the reducing SDS-page electrophoresis, the disulfide bond breaks and the 2 polypeptides form a single band of 20 KDa, which means the protein was a homodimer which had 2 polypeptides made from 20 KDA each bound By disulfide bonds.with another .

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2.Under non-reducing condition, a protein exists as two bands, 30 KDa and 20 KDa. Under reducing conditions, the protein also exists as two bands, 15 KDa and 10 KDa.

Answer: This protein is a Tetramer, consisting for 2 Dimer subunits, bound together non-covalent interactions, in the non-reducing SDS-page electrophoresis these non-covalent bonds break down to form 2 bands of 30 KDA and 20KDA which are the dimer subunits of this protein, in the reducing SDS-page electrophoresis, these dimer subunits dissociate into 4 polypeptide chains because of the breaking of disulfide bonds, causing 2 bands to form 10 KDA which is the 20KDA dimer broken into 2 polypeptides 10 kda each, and 15 KDA which is the 30 KDA dimer broken into 2 polypeptides 15 KDA each.

3.Under non-reducing condition, a protein exists as two bands, 40 KDa and 20 KDa. Under reducing conditions, the protein exists as one bands of 20 KDa.

Answer: This protein is a trimer, Consists of 3 Subunits, a dimer and a monomer, in the non-reducing SDS-page electrophoresis the dimer remains intact because of the covalent disulfide bonds, and the monomer leaves the trimer because it was bound by non-covalent interactions, creating 2 bands a 20KDA band which is the monomer and 40KDA band which is the Dimer, in the reducing SDS-page electrophoresis the dimer is also broken down into polypeptide chains, which creates a single band of 20KDA meaning that the trimer is made of 3 polypeptide chains each of them is 20KDA, 2 of them bound by disulfide bonds and 1 of them is bound to the rest of the protein by non-covalent bonds.