Biotin solution preparation

Add 50 pi of the NHS-LC-biotin solution in DMF to each ml of the protein solution in two aliquots apportioned 10 minutes apart. Alternatively, add a quantity of the sulfo-NHS-biotin solution prepared in water to the protein solution to obtain a 12- to 20-fold molar excess of biotinylation reagent over the quantity of protein present. For instance, for an immunoglobulin (MW 150,000) at a concentration of 10 mg/ml, 20 pi of the sulfo-NHS-biotin solution (8 X 10-4 mmol) should be added per ml of antibody solution to obtain a 12-fold molar excess. 

Add a quantity of the biotin-PEG -amine solution to the solution containing the car-boxylate molecule to achieve the desired molar excess. For molecules containing a single carboxylate to be modified, a 1.5- to 2-fold molar excess may be sufficient. However, for proteins or peptides that also contain competing amines, a much larger excess of biotin compound should be used (e.g., 100-fold excess). For instance, for protein biotinylation, add 120 pi of the biotin-PEG -amine solution per ml of the solution prepared in Step 1. 

Avidin-biotin complex Prepare the ABG according to the instructions provided by the manufacturer and allow the solution to conjugate for at least 30 min at room temperature before applying to slides. Sections should incubate with the ABG for at least 30 min. Rinse sections with buffer. 

To prepare a standard curve, add 0.25 mL of HABA reagent to 10 mL of avidin solution. Incubate 10 min at room temperature and record the absorbance at 500 nm of 1 mL avidin-HABA complex with 0.1 mL buffer, pH 6.0 Distribute 1 mL of the avidin-HABA complex into six test tubes. Add to each the biotin solution in a range of 0 005-0.10 mL. Bring the final volume to 1.10 mL with pH 6.0 buffer, and record the absorbance at 500 nm of each concentration point. Plot a standard curve with the nanomoles of biotin vs the decrease in absorbance at 500 nm. An example of a standard curve is illustrated in Fig 3... 

Immediately before use, dissolve sulfo-NHS-biotin (Pierce) in water at a concentration of 20 mg/ml. Adjust the quantity of this stock solution to be prepared according to the amount of reagent needed to biotinylate the required amount of protein. The sulfo-NHS-biotin solution must be used immediately, since the NHS ester is subject to hydrolysis in aqueous environments. 

Prepare a biotin solution in assay buffer at a final concentration of... 

Biotin labeling Prepare the biotinylation solution shortly before use. Dissolve your protein in PBS (see Note 18). Deliver as much volume of the biotinylation solution to your protein solution as needed to obtain a molar ratio of 20 1 (biotinylation agent protein), i.e., if you have 1 mL of a protein solution at a concentration of 10 pAf, then you need at least 50 jxL of 10 mM biotinylation solution. Let the mixture react for at least 30 min. 

A water-soluble analog of NHS-biotin containing a negatively charged sulfonate group on its NHS ring structure also is available. Sulfo-NHS-biotin may be added directly to aqueous reactions without the need for organic solvent dissolution. A concentrated stock solution may be prepared in water to facilitate the addition of a small quantity to a reaction, but hydrolysis of the NHS ester will occur at a rapid rate, so the solution must be used immediately. 

The NHS ester end of NHS-LC-biotin reacts with amine groups in proteins and other molecules to form stable amide bond derivatives (Figure 11.4). Optimal reaction conditions are at a pH of 7-9, but the higher the pH the greater will be the hydrolysis rate of the ester. Avoid amine-containing buffers which will compete in the acylation reaction. NHS-LC-biotin is insoluble in aqueous reaction conditions and must be solubilized in organic solvent prior to the addition of a small quantity to a buffered reaction. Preparation of concentrated stock solutions may be done in DMF or DMSO. Nonaqueous reactions also may be done with this reagent for the modification of molecules insoluble in water. The molar ratio of NHS-LC-biotin to a... 

Dissolve NHS-LC-biotin (Thermo Fisher) in dry DMF at a concentration of 40 mg/ml. This stock solution is stable for reasonable periods, although long-term storage is not recommended. For use of the water-soluble sulfo-NHS-LC-biotin, a stock solution may be prepared in either organic solvent or water, or the solid reagent may be added directly to the reaction mixture. If a solution in water is made to facilitate the addition of a small quantity of reagent to a reaction, then the solution should be prepared quickly and used immediately to prevent hydrolysis of the NHS ester. Sulfo-NHS-LC-biotin may be dissolved in water at a concentration of 20 mg/ml. 

Biotin-BMCC is insoluble in water and must be dissolved in an organic solvent prior to addition to an aqueous reaction mixture. Preparing a concentrated stock solution in DMF or DMSO allows transfer of a small aliquot to a buffer reaction. The upper limit of biotin-BMCC solubility in DMSO is approximately 33 mM or 17 mg/ml. In DMF, it is only soluble to a level of about 7 mM (4 mg/ml). Upon addition of an organic solution of the reagent to an aqueous environment (do not exceed 10 percent organic solvent in the aqueous medium to prevent protein precipitation), biotin-BMCC may form a micro-emulsion. This is normal and during the course of the reaction, the remainder of the compound will be driven into solution as it couples or hydrolyzes. 

Iodoacetyl-LC-biotin is water-insoluble and therefore must be dissolved in an organic solvent prior to addition to an aqueous reaction medium. Suitable solvents include DMSO and DMF. Concentrated stock solutions may be prepared in DMSO and a small aliquot transferred... 

Prepare a stock solution of the maleimide-PEG -biotin compound in DMAC, DMSO, or DMF (pure and dry solvents only) at a concentration of 10-20 mM. 

Prepare bisulfite modification solution consisting of 3 M concentration of a diamine (i.e., ethylenediamine), 1M sodium bisulfite, pH 6. The use of the dihydrochloride form of the diamine avoids having to adjust the pH down from the severe alkaline pH of the free-base form. Note The optimum pH for transaminating biotin-hydrazide to cytosine residues using bisulfite is 4.5 (see Section 2.3, this chapter). 

A concentrated standard solution of D-biotin, U.S.P. (lmg/ml) was prepared in distilled water a few drops of 1 N KOH were added to... 

Stock solution 4. 100 x stock solution of vitamins was prepared by dissolving biotin (20 mg), folic acid (20 mg), pyrodoxine hydrochloride (100 mg), riboflavin (50 mg), thiamine hydrochloride (50 mg), nicotinic acid (50 mg), pantothenic acid (50 mg), vitamin B12 (1 mg), 4-aminobenzoic acid (50 mg) and thioctic acid (50 mg) in deionized water. The volume was adjusted to 1.0 L. The solution was filtered, sterilized and stored as 10 mL aliquots at —20 °C. 

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