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Biotin and Avidin or streptavidin

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Biotin and Avidin or streptavidin Streptavidin is a 60,000 dalton tetrameric protein purified from Streptomyces avidinii binds very tightly to the vitamin biotin with a Kd of ~10-15 mol/L. This is one of the strongest biological and noncovalent interactions known, and is widely taken advantage of in scientific laboratories.. http://en.wikipedia.org/wiki/Streptavidin

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Biotin and Avidin or streptavidin http://www.arrayit.com/Products/Substrates/SuperStreptavidin/New-Streptavidin-Biotin-Bin.jpg

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Biotin and Avidin or streptavidin http://www.nature.com/nmeth/journal/v3/n4/images/nmeth0406-247-F1.jpg

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Biotin and Avidin or streptavidin http://www.interchim.com/interchim/bio/produits_uptima/images/detection_system.gif

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Biotin and Avidin or streptavidin http://en.wikipedia.org/wiki/Streptavidin Guanidine hydrochloride will dissociate avidin and streptavidin into subunits, but streptavidin is more resistant to dissociation. Streptavidin is much less soluble in water than avidin. In contrast to avidin, streptavidin has no carbohydrate and has a mildly acidic isoelectric point (pI) of ~5. A recombinant form of streptavidin with a mass of 53,000 daltons and a near-neutral pI is commercially available.

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Biotin and Avidin or streptavidin http://en.wikipedia.org/wiki/Streptavidin Because streptavidin lacks any carbohydrate modification and has a near-neutral pI, it has the advantage of much lower nonspecific binding than avidin. A deglycosylated form of avidin, known as NeutrAvidin, also overcomes the nonspecific binding issues with avidin.

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Biotin and Avidin or streptavidin http://en.wikipedia.org/wiki/Streptavidin NeutrAvidin protein is a deglycosylated version of avidin, with a mass of approximately 60,000 daltons. As a result of carbohydrate removal, lectin binding is reduced to undetectable levels, yet biotin binding affinity is retained because the carbohydrate is not necessary for this activity. NeutrAvidin has a near-neutral pI (6.3) that minimizes nonspecific interactions, along with lysine residues that remain available for derivatization or conjugation. Like avidin itself, NeutrAvidin is a tetramer with a strong affinity for biotin (kD = 10-15 M-1).

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Biotin and Avidin or streptavidin http://en.wikipedia.org/wiki/Streptavidin In biochemical applications, streptavidin, which also binds very tightly to Biotin, may be used interchangeably with NeutrAvidin. Avidin immobilized onto solid supports is also used as purification media to capture biotin-labelled protein or nucleic acid molecules. For example, cell surface proteins can be specifically labelled with membrane impermeable biotin reagent, then specifically captured using a NeutrAvidin support

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Biotin and Avidin or streptavidin http://www.piercenet.com

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Protein A and G Protein A and Protein G are the two main proteins with immunoglobulin-binding ability. Their biological function is not yet known. Protein A is a bacterial cell wall protein isolated from Staphylococcus aureus. Protein G is also a cell wall protein obtained from group G Streptococci. While both of these proteins can be used for binding antibodies, compared with protein A, protein G represents a more general and versatile IgG binding reagent [1-3].

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Protein A and G Protein G binds a wider range of IgG subclasses and a greater variety of mammalian species with higher affinity than protein A. In addition, protein G is not as pH dependent as protein A when binding to immunoglobulins [1-3]. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10 [2].

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Protein A and G The equilibrium constants of the reactions between protein G and human, rabbit, mouse and goat polyclonal IgGs ranged between 1 x 1010 and 7 x 1010, for rat polyclonal IgG 1.4 x 109, and human monoclonal IgG1, IgG2, IgG3 and IgG4 between 2 x 109 and 6 x 109. These affinity constants were always greater than the corresponding values for protein A

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Protein A and G (Akerstrom et al., 1985)

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Protein G

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http://www.piercenet.com/Objects/View.cfm?type=Page&ID=75D6C066-AB3E-4B74-87F2-C38A85F0DBC4

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Protein A, G, L

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Protein A, G, L Protein A Protein G Protein A/G Protein L

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Signal Amplification of biosensing systems

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horseradish peroxidase (HRP)

Introduction to liposomes :

Introduction to liposomes Fluorescent nanoparticles Liposomes are spherical nanovesicles consisting of phospholipid bilayers

Introduction to liposomes :

Introduction to liposomes Functional groups for biomolecule conjugation, like sulfhydryl, maleimide and biotin, can be tagged on the liposome surface easily during liposome preparation. Sizes of liposomes can be controlled by extrusion and the diameter for bioassays can be 50 nm, 100nm, 200, 400, and 600 nm.

Liposome encapsulation :

Liposome encapsulation Reverse-phase evaporation method Lipid in organic solvent solution Add nanoparticles in buffer under sonication Evaporation Extrusion Liposomes and unencapsulated nanoparticles Size exclusion chromatography Purified liposomes

Size exclusion chromatography :

Size exclusion chromatography Elution profiles on Sepharose CL-2B column for QDs QD-loaded liposomes were formed and separated Free QDs Free QDs

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Liposomal Nanovesicles (liposomes) Spherical nanovesicles consisting of phospholipid bilayers Several hundred thousand fluorescent dyes

Protein G-immunoliposomes :

Protein G-immunoliposomes Protein G-liposomes

Liposome Preparation :

Liposome Preparation Lipid in organic solvent solution Evaporation Extrusion (or sonication) Liposomes and unencapsulated SRB Lipid film Freeze/thaw cycles Gel filtration Purified liposomes Hydrate with sulforhodamine B (SRB) solution