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BK101

Sigma-Aldrich

ImmunoProbe™ Biotinylation Kit

Synonym(s):

Biotinylation Kits

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About This Item

UNSPSC Code:
41116134
eCl@ss:
34058011
NACRES:
NA.32

storage temp.

2-8°C

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General description

For custom biotinylation of proteins, Sigma offers kits for conjugation on two different scales. The biotin-avidin system has become popular when high sensitivity and specificity are desired. Biotin can be conjugated to antibodies, lectins, enzymes, and other proteins. The protocols in the kits have been optimized for antibodies. Avidin binds to biotin with a high affinity (Ka = 1015 M) and specificity. When conjugated to enzymes or fluorochromes avidin provides a means of identifying biotinylated compounds via enzymatic conversion of substrate to form a visible product or detection of fluorescence by spectrophotometry or flow cytometry.

Application

ImmunoProbe™ Biotinylation Kit has been used for preparation of biotin labeled antibodies.
Biotin has been modified with aminocaproate, then activated via an ester linkage with sulfo-N-hydroxysuccinimide (BAC-Sulfo-NHS). Aminocaproate provides a six-carbon spacer that reduces steric hindrance on the biotin and improves accessibility to the binding site on avidin. Sulfonation of the hydroxysuccinimide increases the polarity of the reagent, allowing it to dissolve easily in aqueous buffer. The ester provides a carbonyl carbon adjacent to a labile ester linkage as a target for primary amine side chains of accessible lysine residues, joining the biotinamidocaproate to the protein via an amide bond.

Features and Benefits

  • Complete protocols for labeling and assay
  • BAC-Sulfo-NHS is completely water soluble
  • No DMF or DMSO needed
  • Biotinylation occurs near neutral pH and physiological ionic strength, avoiding harsh conditions that could damage sensitive proteins
  • Fast separation of conjugate from reactants using gel filtration
  • 2-5 molar ratio biotin to protein in conjugate (for antibodies)
• Two scales to choose from:
1 mg protein per reaction (B-TAG)
10 mg protein per reaction (BK-101)
• Sufficient reagents for at least 5 labelings

Packaging

Kit includes all reagents necessary for biotinylation and for determination of biotin/protein molar ratio.

Analysis Note

Procedure:
Conjugation is performed in four easy steps:
1. Reconstitute BAC-Sulfo-NHS with Phosphate Buffer (PB).
2. Add BAC-Sulfo-NHS to protein and allow to react for 30 minutes at room temperature.
3. Separate the conjugate from the reactants on gel filtration.
4. Assay the conjugate for biotin incorporation by the avidin-HABA assay (BK-101 only). Conjugate is ready to use.

Legal Information

ImmunoProbe is a trademark of Sigma-Aldrich Co. LLC

Kit Components Also Available Separately

Product No.
Description
SDS
  • P38130.01 M PBS, pH 7.4, powder 1 LSDS
  • H51264′-Hydroxyazobenzene-2-carboxylic acid (HABA) 1 mL/vialSDS

Pictograms

Health hazardExclamation mark
GHS08,GHS07

Signal Word

Danger

Hazard Statements

H315,H319,H334,H335

Hazard Classifications

Eye Irrit. 2 - Resp. Sens. 1 - Skin Irrit. 2 - STOT SE 3

Target Organs

Respiratory system

Storage Class Code

10 - Combustible liquids

Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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Hao Chang et al.
Proceedings of the National Academy of Sciences of the United States of America, 109(12), 4455-4460 (2012-03-01)
Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to
Establishment of a new immunological method for direct detection of Mycobacterium in solution
Hamzehlou S and Farajollahi MM
Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi, 49(1), 97-103 (2016)
B Viart et al.
Bioinformatics (Oxford, England), 32(10), 1462-1470 (2016-01-21)
Antibodies are an important class of biological drugs, but with limitations, such as inadequate pharmacokinetics, adverse immunogenicity and high production costs. Synthetic peptides for the desired target represent an important alternative to antibodies. However, no computational tool exists to guide
Does the Sauerbrey equation hold true for binding of peptides and globular proteins to a QCM?: A systematic study of mass dependence of peptide and protein binding with a piezoelectric sensor.
Sohna JES and Cooper MA
Sensing and Bio-Sensing Research, 11, 71-77 (2016)
Mixed self-assembled monolayer (mSAM) based impedimetric immunosensors for cardiac troponin I (cTnI) and soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1)
Billah MDM, et al.
Sensors and Actuators B, Chemical, 173, 361-366 (2012)
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