Types of Chromatography

Ion-exchange

_______ chromatography

• Fractionation based on charge

Gel-filtration / Size Exclusion

_______ chromatography

• Fractionation based on size

Affinity

_______ chromatography

• Fractionation based on binding properties

Anion exchangers

Ion-exchange chromatography:

positively charged resin beads attach to negatively charged molecules

Cation exchangers

Ion-exchange chromatography:

negatively charged resin beads attach to positively charged molecules

large

Gel-filtration chromatography:

• ____ molecules move more rapidly,

small

Gel-filtration chromatography:

• Porous beads attach to _____ molecules

neg

ANION exchangers are ideal for purifying proteins that are _____ charged

pos

CATION exchangers are ideal for purifying proteins that are _____ charged

asp, glu

Ex of amino acids that anion exchangers bind to

lys, arg, his

Ex of amino acids that cation exchangers bind to

Ligand

any molecule that forms specific interactions with a protein.

ligand

Affinity chromatography:

• The ______ is chemically mobilized into a bead, and the sample containing the protein to be purified is applied to the bead

  • Other proteins pass through

metal ions

ligand for affinity tag: Histidine residues

electrophoresis

The migration of the charged molecules in the electric field when an electric field is applies to the solution

cathode

when an electric field is applied to a solution, solute molecules with a net POSITIVE charge migrate towards the ______

anode

when an electric field is applied to a solution, solute molecules with a net NEGATIVE charge migrate towards the ______

supporting medium

electrophoresis should be carried out through some _________

polysaccharide agarose

A gel composed of ______ commonly used for nucleic acids separation

Cross-linked polyacrylamide

______ is commonly used for separating proteins

native gel, SDS-Page, Isoelectric focusing, 2D gel

What are the 4 types of electrophoresis?

native gel

Type of Electrophoresis

• proteins are separated in their native site (no changes)

SDS-Page

Type of Electrophoresis

• SDS detergent is added and therefore proteins are separated based on

  their SIZE differences

Isoelectric focusing

Type of Electrophoresis

• The gel is a pH gradient, and the proteins are separated based on their charge differences ( isoelectric point)

2D gel

Type of Electrophoresis

• SDS-Page & isoelectric focusing are combined

neg

SDS-Page

• Adding SDS causes the protein to carry an overall ____charge that is proportional to the mass of the protein

1, 2

SDS-Page:

• Anions of SDS bind to main chains of polypeptides at a ratio of ____SDS anion for every _____ amino acid residues

denature, elongate

SDS-Page:

• SDS causes a protein to _____ causing it to _____

away

SDS-Page:

• The proteins are put on the opposite side _____ from the positive charge

small

SDS-Page:

• What size of protein travel faster and FURTHER on the gel?

large

SDS-Page:

• What size of protein stays close to the POSITIVE origin?

SDS-Page

_____ allows estimation of the molecular weight of a protein

pos, neg

Isoelectric Focusing:

• The gel is run in an electric field with _____end on the low pH side of the gel and with ____ end on the high pH end.

stops, no

Isoelectric Focusing:

When the pH of the gel region equals pI of a protein, the protein _____ migrating in an electric field because it carries _____ net charge.

keeps, pH = pI

Isoelectric Focusing:

When the pH of the gel region DOESN’T equal pI of a protein, the protein _____ migrating in an electric field till it reaches the region where _____

low

Isoelectric Focusing:

• amino acids with a net NEG charge will migrate towards ____ pH ( POS side)

high

Isoelectric Focusing:

• amino acids with a net POS charge will migrate towards ____ pH ( NEG side)

Isoelectric focusing, SDS-Page

2D Gel / 2D PAGE:

• 1st separation is _____

• 2nd separation is _________

inc, specific activity

how do you know that your purification scheme is working?

• An _____ in _____ after each step of purification indicates that purification is working.

metal ion

If you have a His-tagged protein what would be a good approach of purifying this protein?

• ______ affinity chromatography