Protein Purification and Labeling Techniques: A Comprehensive Guide, Study Guides, Projects, Research of Biochemistry

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MODERN METHODS in

BIOCHEMISTRY

• PROTEIN MODIFICATION
• PROTEIN CROSSLINKING
• PROTEIN STAINING
• ANTIBODY MODIFICATION
• IMMUNOPRECIPITATION
• METABOLIC LABELLING

Affinity chromatography :

• matrices – preparation
• Coupling of Ligand
• detection of reactive groups

Mouse brain

Solubilise & homogenise

50,000 x g

supernatant

Affinity Chromatography

Immunoprecipitation

eluted complexes

Gel filtration

SDS-PAGE

Affinity chromatography Example

Affinity

chromatography

matrice preparation & introduce spacer

Affinity chromatography Example

COMMONLY USED AGENTS

1. Cyanogen bromide

• Cyanogen bromide (CNBr) is one of the most widely

used linkers in affinity chromatography. It reacts with

hydroxyl groups in agarose and other polysaccharide

materices to produce a reactive linker. The linker can

be connected to ligands or spacers which contain

primary amine groups (such as proteins).

• Cyanogen bromide is extremely toxic as it releases

hydrogen cyanide on acidification.

COMMONLY USED AGENTS

3. Carbonyldiimidazole

• N,N'carbonyldiimidazole (CDI) is reacts with

polysaccharides to form imidazole carbonate

derivatives. These derivatives then react with ligands

containing primary amino groups at an alkine pH to

give stable carbonate derivatives.

4. Sulphonyl chloride

• Reacts with hydroxyl groups on the matrix to

form sulphonyl esters. These esters react with amino

and thiol groups of the ligand.

5. Sodium periodate (NaIO4)

• Reacts with diol groups on polysaccharide matrices to

form reactive aldehydes. These react with primary amines

to form Schiff's bases.The Schiff's bases can be stabilized

by reduction with sodium borohydride.

• Periodate is easy to use and non-toxic. The product is

stable.

6. Glutaraldehyde

• Activates amino or amide groups on polyacrylamide

matrices or on agarose with amine spacers. These groups

can then react with primary amine groups on the ligand.

Brand Name Ligand Application Examples

HiTrap Protein A Protein A IgG, IgG subclasses, fragments of IgG, IgA, antigens, immune complexes HiTrap Progein G Recombinant Protein G IgG, IgG subclasses lacking the albumin binding region HiTrap Heparin Heparin Growth factors, coagulation proteins, plasma proteins, lipases, lipoproteins, enzymes that act on nucleic acids, steroid receptors, protein synthesis factors HiTrap Chelating Iminodiacetic acid Proteins and peptides with exposed histidine groups HiTrap Blue Cibacron Blue F3GA Enzymes requiring adenyl-containing cofactors, albumin, coagulation factors, interferon

Brand Name Ligand Applications

Affi-Gel Protein A Protein A Purification of IgG from acites, serum and Affi-Prep Protein A culture fluid. Affi-Gel Blue Cibacron Blue F3GA Binds many nucleotide-requiring enzymes, albumin and other proteins. DEAE Affi-Gel Blue Cibacron Blue F3GA Binds albumin and serum proteins and DEAE groups. Used to purify protease-free IgG from acites, serum and cutlure fluid Affi-Gel heparin Heparin Purification of a wide variety of proteins including growth factors, coagulation factors, DNA and RNA specific enzymes, lipase, lipoproteins and proteases Affi-Prep polymyxin Polymixin Endotoxin removal Affi-Gel 501 Organomercurial Adsorbtion of sulfhydrl proteins and low mw sulfhydrls via thiol groups. Bound proteins are eluted with dilute mercaptoethanol or dithioreitol. Affi-Gel 601 Boronate Adsorbtion of cis-hyroxyl containing molecules including sugars, nucleotides and glycocpeptides.

PROTEIN STAINING

PROTEIN STAINING

• Amidoblack
• Commassie
• Ponceau-red
• Silver
• Gold
• Gelcode

Silver-stained two-dimensional polyacrylamide-gel

PROTEIN STAINING Amidoblack

spot 5 ul protein sample on nitrocellulose (NC)

• air-dry for 5 min
• immerse in stain solution for 3 min
• wash 2x 3 min in water
• wash 2x 3 min in wash solution
• wash 5 min with water
• air-dry 5 min
• elute stain with 1 ml elution solution (while shaking the sample)
• measure absorption at 630 nm of the eluant