Bissulfosuccinimidyl suberate: Difference between revisions
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{{Cleanup|date=May 2007}} |
{{Cleanup|date=May 2007}} |
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== Introduction to |
== Introduction to Crosslinkers == |
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Crosslinkers are chemical reagents that play a crucial role in the preparation of conjugates used in biological research particularly immuno-technologies and protein studies. Crosslinkers are designed to covalently interact with molecules of interest, resulting in conjugation. A [[spacer arm]], generally consisting of several atoms, separates the two molecules, and the nature and length of this spacer is important to consider when designing an assay involving the selected crosslinker. BisSulfosuccinimidyl suberate is an example of a homobifunctional crosslinker. |
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== BisSulfosuccinimidyl Suberate: General Information == |
== BisSulfosuccinimidyl Suberate: General Information == |
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'''Abbreviations:''' (BS3), Sulfo-DSS |
'''Abbreviations:''' (BS3), Sulfo-DSS |
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'''Molecular formula:''' |
'''Molecular formula:''' C<sub>16</sub>H<sub>18</sub>N<sub>2</sub>O<sub>14</sub>S<sub>2</sub>Na<sub>2</sub> |
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'''Molecular weight:''' 572.43 |
'''Molecular weight:''' 572.43 |
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== BS3 Characteristics == |
== BS3 Characteristics == |
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'''Water soluble:''' BS3 is hydrophilic due to its terminal sulfonyl substituents and as a result dissociates in water, eliminating the need to use organic solvents which interfere with protein structure and function. Because organic solvents need not be used when BS3 is used as the |
'''Water soluble:''' BS3 is hydrophilic due to its terminal sulfonyl substituents and as a result dissociates in water, eliminating the need to use organic solvents which interfere with protein structure and function. Because organic solvents need not be used when BS3 is used as the crosslinker, it is ideal for investigations into protein structure and function in physiologic conditions. |
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'''Non-cleavable:''' BS3 binds irreversibly to its conjugate molecules, meaning that once BS3 creates covalent linkages to its target molecules, those associations are not easily broken |
'''Non-cleavable:''' BS3 binds irreversibly to its conjugate molecules, meaning that once BS3 creates covalent linkages to its target molecules, those associations are not easily broken. |
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'''Membrane impermeable:''' Since BS3 is a charged molecule, it cannot freely pass through cellular membranes which makes it an ideal |
'''Membrane impermeable:''' Since BS3 is a charged molecule, it cannot freely pass through cellular membranes which makes it an ideal crosslinker for cell surface proteins. |
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'''Homobifunctional:''' BS3 is a homobifunctional crosslinker in that is has two identical reactive groups, i.e. the N-hydroxysulfosuccinimide esters, and only one step is necessary to establish |
'''Homobifunctional:''' BS3 is a homobifunctional crosslinker in that is has two identical reactive groups, i.e. the N-hydroxysulfosuccinimide esters, and only one step is necessary to establish crosslinking between conjugate molecules. |
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'''Amine reactive:''' BS3 is amine |
'''Amine reactive:''' BS3 is amine-reactive in that its N-hydroxysulfosuccinimide (NHS) esters at each end react specifically with primary amines to form stable amide bonds in an SN2-type reaction in which the N-hydroxysulfosuccinimide acts as the leaving group. BS3 is particularly useful in protein-related applications in that it can react with the primary amines on the side chain of lysine residues and the N-terminus of polypeptide chains. This crosslinker can also be used to stabilize protein-protein interactions for further analysis by immunoprecipitation. |
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== DiSuccinimidyl Suberate: non-water soluble analog of BS3 == |
== DiSuccinimidyl Suberate: non-water soluble analog of BS3 == |
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DiSuccinimidyl Suberate, DSS, is the non-water soluble analog of BS3. |
DiSuccinimidyl Suberate, DSS, is the non-water soluble analog of BS3. DSS and BS3 express the same crosslinking ability toward primary amines. The major structural difference between these two molecules is that DSS does not contain the sulfonyl substituents at either end of the molecule, and it is this difference that is responsible for the uncharged, non-polar nature of the DSS molecule. Due to the hydrophobic nature of this crosslinker it must be dissolved in an organic solvent such as dimethylsulfoxide before being added to an aqueous sample. Because of the ability of DSS to cross cell membranes, it is best suited for applications where intracelluclar crosslinking is needed. |
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[[Image:DSS_struct.gif]] |
[[Image:DSS_struct.gif]] |
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== General Applications == |
== General Applications == |
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1) |
1) cell-surface receptor-ligand studies; |
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2) |
2) crosslinking biomolecules on cells; |
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3) fixation of protein complexes prior to protein interaction analysis |
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== Standard Protocol for Use of BS3: Adapted from Uptima == |
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This standard protocol describes the coupling attachment of IgG onto a protein A agarose gel, and should be optimized to each application (volume, type of Ig and gel…).<ref>http://www.interchim.com/interchim/bio/produits_uptima/tech_sheet/FT-UP28065(DSS,BS3).pdf</ref> For applications from 1 to 10 mg of IgG, 1 mL of gel is usually needed (pipette a 2mLof 50% suspension). |
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Incubations and washes are performed in batches of 5 mL buffer per 1 mL gel. |
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1) Generate an agarose protein A gel. Wash well with PBS (150mM NaCl, 20mM phosphate, pH7.4) |
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2) Incubate the antibody in the appropriate quantity onto Protein A for 30 minutes at room temperature (25 degrees C) under constant and gentle agitation. |
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3) Wash well with PBS. Absorbance at 280nm should be less than 0.05. |
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N.B.: the unbound antibody can be recovered in washes and quantified by a protein assay to determine the loading capacity of the gel, by difference with the initially added IgG. |
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4) Prepare a DSS solution at 20 mM in anhydrous DMSO. This solution should be used immediately. The BS3 is dissolved at 20 mM in aqueous buffer. |
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5) Add very slowly 175μl of DSS solution or BS3 solution per mLof gel in 2mL of PBS while stirring. Complete in 2 or 3 steps separated by 5 min intervals. Incubate for 1 hour under constant agitation at room temperature. |
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6) Wash the gel with PBS (at least 3 washes of 5 mL buffer per 1 mLof gel over a 5 minute interval) |
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7) Incubate the gel with a saturating agent, 5 mL buffer/mL of gel for 1 hour at room temperature. |
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== Specific Application: BS3 application in biomedical research == |
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Effect of Ischemic Preconditioning on the Surface Expression of NMDA Receptors in Hippocampal Neurons. |
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Excitotoxic death of central neurons is implicated in neurodegeneration after stroke, trauma and several other neurological diseases. Typically, a neurological trauma initially causes oxygen-glucose deprivation (OGD); the cells, deprived of oxygen and glucose, are unable to perform aerobic or anaerobic respiration. If severe enough, OGD results in neuronal death, and lysed neuron bodies release a flood of neurotransmitters previously contained within the cell membrane. So while cerebral neurons in the region most severely affected by hypoxia or anoxia die rapidly by necrosis, the majority of neurons that die in the surrounding tissue die by apoptosis induced by overexcitation; a sort of delayed cell death following the initial anoxia induced necrosis. Largely mediated by over-activation of calcium-permeable NMDA receptors (NMDA-Rs), excitotoxicity leads to excessive Ca2+ entry and cellular Ca2+ overload. Following such an NMDA-R stress, a Ca2+ precipitate forms within the mitochondria indicating the role of sequestration as a buffering mechanism; maintaining relatively low levels of free Ca2+ in the cellular matrix. Ca2+ is one of several pro-apoptic second messengers causing mitochondrial membrane permeabilization (MMP) that leads to the release of proteases and nuclease activators normally confined to the mitochondrial intermembrane space. Studies of patient tissues and animal models have also shown that mitochondria-mediated apoptosis kills many neurons after an acute stroke or traumatic injury (Kroemer et al. 2003). Therefore, the reduction of this Ca2+ load has been interpreted as a critical neuroprotective aspect of preconditioning (PC). |
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PC is the phenomenon whereby neurons become resistant to a normally lethal excitotoxic insult if they have been pretreated by exposure to a similar but milder, non-toxic insult. The Andrews lab had previously shown that highly effective PC-induced neuroprotection of hippocampal neurons is paralleled by the reduction of evoked cytosolic Ca2+ elevations as well as increased Ca2+ concentration capacity. This suggests that strong PC involves a change in NMDA-R activity or availability. A role for the NMDA receptor has also been suggested by Kato et al. (1992), Kasischke et al. (1996), and Heurteaux et al. (1995). The lab of Dr. Andrews (NINDS) are interested in the mechanism of PC as one working through this NMDA pathway. During my summer internship (2006) in this lab, we tested the hypothesis that one specific mechanism of PC is a decrease in the surface expression of functional NMDA-Rs, thereby leading to reduced Ca2+ entry and reduced cell death.Before harvest, we treated intact hippocampal cultures conditioned and unpreconditioned with chymotrypsin or with the crosslinking reagent BS3 for 10min. This technique was utilized in Dr. Soderling’s research at the Vollum Institute in determining differential surface expression of NMDA-R subunits NR1 and NR2. The protease chymotrypsin cleaves the extracellular domains of plasma membrane proteins, including the NR1 subunit of NMDA-Rs. BS3 connects NR-1 subunits to each other and associated proteins. Following the treatment, the cultures were washed and harvested in ice-cold harvest buffer; the components of which inhibit any unreacted chymotrypsin or BS3. |
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Following Invitrogen protocols, we then ran 2 SDS-PAGE gels (Invitrogen): one for Silver Stain (Invitrogen) and the other for western blotting (Invitrogen) and Blue Stain (Invitrogen). Lanes contained samples of unpreconditioned control, chymotrypsin, and BS3 treated cultures and their preconditioned corollaries. Monoclonal Chemicon anti-NR1 was used for Western blot staining. The comparison of particular interest to our research was between the ratio of intracellular to extracellular expressed NR1 in nonpreconditioned cells to preconditioned cells. |
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Densitometry on western blots developed with an NR1-specific antibody (Chemicon) was used to quantify changes in surface expression of NMDA-Rs as a result of preconditioning. Using the ImageJ program, we created histograms of chromagen stained western blot lanes. Drawing base lines under peaks representing NR1 bands, ImageJ calculated the area within these bound regions and percentage ratios for samples treated with chymotrypsin and BS3. This enabled us to compare the percentage of intracellular to extracellular NR1 in naïve cells to preconditioned cells. |
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For greater sensitivity to the NR1 protein, we decided to utilize chemiluminescence (Invitrogen) that is capable of measuring picogram amounts. |
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== Results == |
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Western blotting of control samples showed an overall reduction in intensity of the major NR1 band in PC cells compared to noPC cells. Samples treated with either proteolysis or the cross-linking reagent revealed patterns of staining representing cleavage or cross-linking, respectively, of surface-exposed NMDA-NR1 subunits. Chymotrypsin treated samples had lighter molecular weight breakdown products less than 100kDa revealed as diffuse 51-64kD bands corresponding to fragments of the surface expressed receptor, as well as a 100kD band corresponding to the intact intracellular NR1 pool. BS3 gave similar results, as revealed by the appearance of high molecular weight, aggregated species representing NMDA-subunits cross-linked together or with other related surface proteins. Typically, 50% of NR1 was surface expressed. |
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Western blot densitometry analysis of NR1 distribution, as obtained by chymotrypsin or cross-linker BS3, reveals significant reduction in NR1 surface expression after preconditioning. Thus, the intracellular pool of NR1 was found to be increased 9% (for chymotrypsin) and 14% (for BS3) in PC cells as compared with naive cells. |
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[[Image:asym.jpg]] |
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BS3 covalently bonds to primary amines of lysine side chains and N-terminus polypeptide chains; effectively cross-linking NR-1 subunits and related proteins. |
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== References == |
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<references/> |
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* Heurteaux C, Lauritzen I, Widmann C, Lazdunski M (1995) Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditionining. Proc Natl Acad Sci USA 92:4666-4670. |
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* Kasischke K, Ludoplph AC, Riepe MW (1996) NMDA antagonists reverse increased hypoxic tolerance by preceding chemical hypoxia. Neurosci Lett 214:175-178. |
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* Kato H, Liu Y, Araki T, Kogure K (1992) MK-801, but not anisomycin, inhibits the induction of tolerance to ischemica in the gerbil hippocampus. Neurosci Lett 139:118-121. |
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* http://www.rcsb.org/pdb/explore.do?structureId=1PB9 Accessed: April 16, 2007. Released: 2003-06-24. CRYSTAL STRUCTURE OF THE NR1 LIGAND BINDING CORE IN COMPLEX WITH D-CYCLOSERINE AT 1.60 ANGSTROMS RESOLUTION. Authors: Fukawa, H., Gouaux, E. |
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* http://www.rcsb.org/pdb/explore.do?structureId=2NR1 Accessed: April 16, 2007. Released: 1998-04-29 TRANSMEMBRANE SEGMENT 2 OF NMDA RECEPTOR NR1, NMR, 10 STRUCTURES Authors: Gesell, J.J., Sun, W., Montal, M., Opella, S. |
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* http://www.rcsb.org/pdb/explore.do?structureId=1PB9 Accessed: April 16, 2007. Furukawa, H., Gouaux, E. Mechanisms of activation, inhibition and specificity: crystal structures of the NMDA receptor NR1 ligand-binding core, Embo Journal, Vol.22, pp.2873-2885, 2003. |
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[[Category:Molecular biology]] |
[[Category:Molecular biology]] |
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Revision as of 17:15, 25 September 2007
 |
Introduction to Crosslinkers
Crosslinkers are chemical reagents that play a crucial role in the preparation of conjugates used in biological research particularly immuno-technologies and protein studies. Crosslinkers are designed to covalently interact with molecules of interest, resulting in conjugation. A spacer arm, generally consisting of several atoms, separates the two molecules, and the nature and length of this spacer is important to consider when designing an assay involving the selected crosslinker. BisSulfosuccinimidyl suberate is an example of a homobifunctional crosslinker.
BisSulfosuccinimidyl Suberate: General Information
Abbreviations: (BS3), Sulfo-DSS
Molecular formula: C16H18N2O14S2Na2
Molecular weight: 572.43
Spacer arm length: 11.4 Å (8 atoms)
CAS Number: 82436-77-9
Storage conditions: 4°C, protect from moisture, use only fresh solutions
BS3 Characteristics
Water soluble: BS3 is hydrophilic due to its terminal sulfonyl substituents and as a result dissociates in water, eliminating the need to use organic solvents which interfere with protein structure and function. Because organic solvents need not be used when BS3 is used as the crosslinker, it is ideal for investigations into protein structure and function in physiologic conditions.
Non-cleavable: BS3 binds irreversibly to its conjugate molecules, meaning that once BS3 creates covalent linkages to its target molecules, those associations are not easily broken.
Membrane impermeable: Since BS3 is a charged molecule, it cannot freely pass through cellular membranes which makes it an ideal crosslinker for cell surface proteins.
Homobifunctional: BS3 is a homobifunctional crosslinker in that is has two identical reactive groups, i.e. the N-hydroxysulfosuccinimide esters, and only one step is necessary to establish crosslinking between conjugate molecules.
Amine reactive: BS3 is amine-reactive in that its N-hydroxysulfosuccinimide (NHS) esters at each end react specifically with primary amines to form stable amide bonds in an SN2-type reaction in which the N-hydroxysulfosuccinimide acts as the leaving group. BS3 is particularly useful in protein-related applications in that it can react with the primary amines on the side chain of lysine residues and the N-terminus of polypeptide chains. This crosslinker can also be used to stabilize protein-protein interactions for further analysis by immunoprecipitation.
DiSuccinimidyl Suberate: non-water soluble analog of BS3
DiSuccinimidyl Suberate, DSS, is the non-water soluble analog of BS3. DSS and BS3 express the same crosslinking ability toward primary amines. The major structural difference between these two molecules is that DSS does not contain the sulfonyl substituents at either end of the molecule, and it is this difference that is responsible for the uncharged, non-polar nature of the DSS molecule. Due to the hydrophobic nature of this crosslinker it must be dissolved in an organic solvent such as dimethylsulfoxide before being added to an aqueous sample. Because of the ability of DSS to cross cell membranes, it is best suited for applications where intracelluclar crosslinking is needed.
General Applications
1) cell-surface receptor-ligand studies; 2) crosslinking biomolecules on cells; 3) fixation of protein complexes prior to protein interaction analysis