INSTRUCTIONS
Number Description
21335 EZ-Link® Sulfo-NHS-LC-Biotin, 100 mg
21327 EZ-Link Sulfo-NHS-LC-Biotin, No-Weigh™ Format, 8 × 1 mg microtubes Molecular Weight: 556.59
Spacer Arm: 22.4 Å
O O
H
N
O
NH
HN
S
O
N O
S O
O
气功养生O Na
Storage: Upon receipt store desiccated at -20°C. EZ-Link Sulfo-NHS-LC-Biotin is shipped at ambient
temperature. No-Weigh Format Sulfo-NHS-LC-Biotin is shipped with an ice pack.
Table of Contents Introduction (1)
Important Product Information (2)
Additional Materials Required (2)
六年级作文大全Procedure for Biotinylating Proteins in Solution (2)
Procedure for Biotinylating Cell Surface Proteins (3)
Related Thermo Scientific Products (4)
Cited References (4)
Product References (4)
Introduction
Sulfo-NHS-LC-Biotin (sulfosuccinimidyl-6-[biotin-amido]hexanoate) enables simple and efficient label
ing of antibodies, proteins and any other primary amine-containing molecules. Specific labeling of cell surface proteins is another common application for this water-soluble and membrane impermeable reagent. The No-Weigh Format Sulfo-NHS-LC-Biotin is consists of convenient single-u microtubes, eliminating difficulties associated with weighing small quantities of reagent. Biotin is a vitamin that binds with high affinity to avidin and streptavidin proteins. Becau it is small (244 Da), biotin can be conjugated to many proteins without altering their biological activities. Labeled proteins can be purified from unlabeled proteins using immobilized streptavidin and avidin (e Related Products) and detected in ELISA, dot blot or Western blot applications using streptavidin or avidin-conjugated probes.
N-Hydroxysuccinimide (NHS) esters of biotin are the most popular type of biotinylation reagent. NHS-activated biotins react efficiently with primary amino groups (-NH2) in pH 7-9 buffers to form stable amide bonds. Proteins, including antibodies, generally have veral primary amines in the side chain of lysine (K) residues and the N-terminus of each polypeptide that are available as targets for labeling with NHS-activated biotin reagents. Several different NHS esters of biotin are available, with varying properties and spacer arm lengths. Sulfo-NHS-LC-Biotin is water soluble, enabling reactions to be performed in the abnce of organic solvents such as DMSO or DMF.
Cell-surface biotinylation has emerged as an important tool for studying the expression and regulation of receptors and transporters, differentiation of plasma membrane proteins from tho localized to organelle membranes, and distribution of membrane proteins in polarized epithelial cells. The specificity of Sulfo-NHS-LC-Biotin for cell-surface labeling has been demonstrated in the applications.1,2 Becau Sulfo-NHS-LC-Biotin dissolves readily in polar solutions and is charged by the sodium sulfoxide group on the succinimidyl ring, it cannot permeate the cell membrane. As long as the cell remains intact, only primary amines expod on the surface will be biotinylated.
Sulfo-NHS-LC-Biotin
Important Product Information
• Sulfo-NHS-LC-Biotin is moisture-nsitive. Store the vial of biotin reagent at -20°C with desiccant. To avoid moisture condensation onto the product, equilibrate vial to room temperature before opening.
•
No-Weigh Format Handling: Immediately before u, puncture the microtube foil with a pipette tip, add water and mix by pipetting up and down. After u, cut the ud microtube from the microtube strip and discard. Store the unud microtubes in the foil pouch provided.
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As directed in the procedure, dissolve the biotin reagent immediately before u. The NHS-ester moiety readily hydrolyzes and becomes non-reactive; therefore, do not prepare stock solutions for storage. Discard any unud reconstituted reagent.
•
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Avoid buffers containing primary amines (e.g., Tris or glycine) as the will compete with the intended reaction. If necessary, dialyze or otherwi desalt to exchange the protein sample into an amine-free buffer such as phosphate-buffered saline (e Related Products).
•
When biotinylating proteins in solution, excess non-reacted biotin and reaction byproducts are easily removed by size exclusion using either desalting columns or dialysis. A 10 ml desalting column is best suited for processing reactions involving 1-10 mg of protein in approximately 0.5-2 ml. For smaller protein amounts or reaction volumes, both the
biotinylation reaction and subquent buffer exchange can be performed in a single Slide-A-Lyzer ® MINI Dialysis Unit. For reaction volumes too large for processing with a desalting column, either split the sample between two columns or u an appropriate Slide-A-Lyzer Dialysis Castte. For processing small volumes (i.e., 10-150 µl) of peptides and other low molecular weight molecules, Pierce ® C-18 Spin Columns (Product No. 89870 or 89873) may be ud.
Additional Materials Required
• Phosphate-buffered saline (PBS) or other amine-free buffer having pH 7-8 for u as reaction buffer (e Important Product Information and Related Products)
• Desalting columns or dialysis units for buffer exchange (e Important Product Information and Related Products)
Procedure for Biotinylating Proteins in Solution
A. Calculations
The extent of biotin labeling depends on the distribution of amino groups on the protein, protein concentration and the amount of reagent ud. Compared to reactions involving concentrated protein solutions, labeling reactions with dilute solutions require a greater fold molar excess of biotin reagent to achieve the same incorporation level. Experiments
performed at Pierce that ud a 20-fold molar excess of biotin reagent to label 1-10 mg antibody (in 0.5-2 ml) resulted in
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4-6 biotin groups per antibody molecule. Experiments that ud a 50-fold molar excess of biotin reagent to label 50-200 µg of antibody (in 200-700 µl) resulted in 1-3 biotin groups per antibody molecule. Adjust the molar ratio of Sulfo-NHS-LC-Biotin to protein to obtain the level of incorporation desired.
1. Calculate millimoles of biotin reagent to add to the reaction for a 20-fold molar excess:
分别财产制
Biotin mmol protein
mmol Biotin
mmol 20 protein mg protein mmol protein ml protein mg protein ml =×××
• 20 = Molar fold excess of biotin
2. Calculate microliters of 10 mM biotin reagent solution (prepared in Step B.3) to add to the reaction:
Biotin µl mmol
10L
L µl 1,000,000Biotin mmol =××
B.Biotin Labeling Reaction
1.Remove vial of Sulfo-NHS-LC-Biotin from freezer and equilibrate it to room temperature before opening in Step 3.
2.Prepare protein in PBS according to the calculation made in Section A.
Note: Protein that is already dissolved in amine-free buffer at pH 7.2-8.0 may be ud without buffer exchange or dilution with PBS. Proteins in Tris or other amine-containing buffers must be exchanged into a suitable buffer.
3.Immediately before u, prepare a 10 mM solution of the biotin reagent:
•Product No. 21335 (100 mg vial): Add 360 µl of ultrapure water to 2.0 mg of reagent.
•Product No. 21327 (No-Weigh Format): Add 180 µl of ultrapure water to the 1 mg microtube.
4.Add the appropriate volume (e Calculations in Section A) of 10 mM biotin reagent solution to the protein solution.
5.Incubate reaction on ice for two hours or at room temperature for 30 minutes.
Note: Other than the possibility of ordinary protein degradation or microbial growth, there is no harm in reacting longer than the specified time.
6.Protein labeling is complete at this point, and although excess non-reacted and hydrolyzed biotin reagent remains in the
solution, it is often possible to perform preliminary tests of the labeled protein by ELISA or Western blot. Once proper function and labeling of the protein has been confirmed, for optimal performance and stability, purify the labeled protein using desalting or dialysis. If the Biotin Quantitation Kit (HABA assay; Product No. 28005) will be performed to determine the level of biotin incorporation, the protein first must be desalted or dialyzed to remove non-reacted biotin.
Procedure for Biotinylating Cell Surface Proteins
Many variations of this procedure exist in the literature (e Product References). Labeling may be performed on cells in suspension or on adherent cells in culture plates. In the latter situation, diffusion of the Sulfo-NHS-LC-Biotin to all surfaces of the cells will be limited, and labeling will occur predominately on the expod surface. Culture media must be washed from cells; otherwi, amine-containing components will compete and quench the reaction to cell surface proteins. Using a more
concentrated cell suspension is most effective becau less biotin reagent is required in the reaction. Generally, a final concentration of 2-5 mM Biotin Reagent is effective. NHS reactions occur more rapidly at high pH; therefore, pH 8.0 is ud in the following example so that labeling can be completed as quickly as possible.
1.Wash cells three times with ice-cold PBS (pH 8.0) to remove amine-containing media and proteins from the cells.
2.Suspend cells at a concentration of ~25 × 106 cells/ml in PBS (pH 8.0).
3.Add 1.0 mg of Sulfo-NHS-LC-Biotin reagent per ml of cell suspension (results in ~2 mM biotin reagent). Alternatively,
add 200 µl of the 10 mM biotin reagent solution (e step B.3 on previous page) per ml of cell suspension.
4.Incubate reaction mixture at room temperature for 30 minutes.
Note: Performing this incubation at 4°C may reduce active internalization of the biotin reagent.
5.Wash cells three times with PBS + 100 mM glycine to quench and remove excess biotin reagent and byproducts.
Additional Information Available on the Web
•Tech Tip #14: Perform labeling and other reactions in Slide-A-Lyzer Dialysis Casttes
•Tech Tip #43: Protein stability and storage
•HABA Calculator for computing the results associated with the HABA assay measurements
Related Thermo Scientific Products
28372 BupH™ Phosphate Buffered Saline Packs, 40 pack, each pack yields 500 ml of 0.1 M sodium phosphate, 0.15 M sodium chloride, pH 7.2 when reconstituted with 500 ml water.
69576 Slide-A-Lyzer MINI Dialysis Unit Kit, for 10-100 µl sample volumes, 10 units plus float
因为你英文66382, 66807 Slide-A-Lyzer Dialysis Castte Kits, for 0.5-3 ml and 3-12 ml sample volumes, respectively 89892 Zeba Desalt Spin Columns, 5 ml, 25 columns, for 500-2,000 µl samples
28005 EZ Biotin Quantitation Kit, HABA assay kit to determine levels of biotin incorporation
Agaro, 2 ml
20347 Streptavidin
20228 Monomeric Avidin Agaro Kit, bind and gently elute biotin-labeled molecules
Horradish Peroxida Conjugated, 1 mg
21126 Streptavidin,
21445 EZ-Link Sulfo-NHS-SS-Biotinylation Kit, ~10 reactions with 1-10 mg of protein
21935 EZ-Link Micro Sulfo-NHS-LC-Biotinylation Kit, 8 reactions with 50-200 µg of protein
89870 Pierce C-18 Spin Columns, 25 columns
Cited References
1.Daniels, G.M. and Amara, S.G. (1998). Selective labeling of neurotransmitter transporters at the cell surface. Methods Enzymol.296:307-18.
2.Huh, K-H. and Wenthold, R.J. (1999). Turnover analysis of glutamate receptors identifies a rapidly degraded pool of the N-methyl-D-aspartate receptor
subunit, NR1, in cultured cerebellar granule cells. J. Biol. Chem. 274:151-7.
Product References
Ali, M.K. and Bergson, C. (2003). Elevated intracellular calcium triggers recruitment of the receptor cross-talk accessory protein calcyon to the plasma membrane. J. Biol. Chem.278(51):51654-63.
Borroto, A., et al. (2003). Impaired trafficking and activation of tumor necrosis factor-α-converting enzyme in cell mutants defective in protein ectodomain shedding. J. Biol. Chem.278(28): 25933-9.
Chyung, J.H. and Selkoe, D.J. (2003). Inhibition of receptor-mediated endocytosis demonstrates generation of amyloid β-protein at the cell surface. J. Biol.
Chem.278(51):51035-43.
Frickel, E-M., et al. (2002). TROSY-NMR reveals interaction between Erp57 and the tip of the calreticulin P-domain. PNAS99(4):1954-9.
Gimferrer, I., et al. (2003). The accessory molecules CD5 and CD6 associate on the membrane of lymphoid T cells. J. Biol. Chem.278(10):8564-71.
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Li, H. and Pajor, A.M. (2003). Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity. Am. J. Physiol. Cell Physiol. 285:C1188-96. Lukashevich, I.S., et al. (2003). Arenavirus-mediated liver pathology: acute lymphocytic choriomeningitis virus infection of rhesus macaques is characterized by high-level interleukin-6 expression and hepatocyte proliferation. J. Virol.77(3):1727-37.
Ohnishi, T., et al. (2003). MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation esntial for its translocation to the cell surface.
Clin. Diagn. Lab. Immunol.10(3):405-10.
Sulfo-NHS Technology is protected by U.S. Patent #s 6,407,263, 5,872,261, 5,892,057 and 5,942,628.
Slide-A-Lyzer® Dialysis Castte Technology is protected by U.S. Patent # 5,503,741 and 7,056,440.
Slide-A-Lyzer® MINI Dialysis Unit Technology is protected by U.S. Patent # 6,039,871.
This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as t forth in the Product documentation, specifications and/or accompanying package inrts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwi expressly authorized in writing, Products are supplied for rearch u only. No claim of suitability for u in applications regulated by FDA is made. The warranty provided herein is valid only when ud by properly trained individuals. Unless otherwi stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchar of the Product (“Buyer”).
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