Level of Biotinylation

Chapter 8, section 3, describes the major biotinylation compounds and their properties. Also provided in that section are suggested protocols for reacting each of these reagents with specific functional groups on macromolecules. 

Since a biotinylated molecule is able to interact with avidin at its biotin binding sites just as strongly as biotin in solution, the degree of biotinylation may be determined using the HABA method as well. Comparison of the response of a biotinylated protein, for example, with a standard curve of various biotin concentrations allows calculation of the molar ratio of biotin incorporation. 

The following protocol describes both of these HABA-based tests for determining the level of biotinylation. 

Dissolve avidin in 0.05 M sodium phosphate, 0.15 M NaCl, pH 6, at a concentration of 0.5 mg/ml. A total of 3 ml of the avidin solution is required to create a standard curve using known concentrations of biotin and an additional 3 ml is needed for each sample determination. 

Dissolve the HABA dye (Sigma) in 10 mM NaOH at a concentration of 2.42 mg/ml (10 mM). Prepare about 100 pi of the HABA solution for each 3-ml portion of avidin solution required. 

It is often important to determine the extent of biotin modification after a biotinylation reaction is complete. Measuring biotin incorporation into macromolecules can aid in optimizing a particular (strept)avidin-biotin assay system. It also can be used to assure reproducibility in 

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Dissolve an antibody or protein to be modified at a concentration of l-10mg/ml in 0.1 M sodium phosphate, 0.15M NaCl, pH 1.2-1.5. Lower concentrations of protein may result in decreased reaction yields and require increased quantities of reagent to obtain acceptable levels of biotinylation. Avoid amine-containing buffers or components, such as Tris or imidazole, which will react with the NHS ester and interfere with the biotinylation process. 

Add a 3-fold molar excess of biotinylation reagent over the molar quantity of dendrimer present. For the use of sulfo-NHS-LC-biotin (MW 556), this represents the addition of 2.1pmol or 1.16mg. This reaction ratio will result in a modification level of about 2.5 biotin groups per dendrimer. Other molar ratios also may be used, depending on the desired level of modification and the intended use for the conjugate. 

For instance, a dendrimer easily can be coupled with a large number of fluorescent dyes and still provide additional coupling sites for biotinylation. The only limitation to the number of fluorescent modifications is if fluorescence quenching starts to take place, in which case no further modifications will result in increased signal. A series of such conjugates using different levels of fluorophore modification should be done to determine the optimal level of dye-to-dendrimer before quenching occurs. 

Add 0.3 mg of sulfo-NHS-SS-biotin (Thermo Fisher) to each ml of the antibody solution. To measure out small amounts of the biotinylation reagent, it may be first dissolved in water at a concentration of at least 1 mg/ml. Immediately transfer the appropriate amount to the antibody solution. This level of sulfo-NHS-SS-biotin addition represents about an 8-fold molar excess over the amount of antibody present. This should result in a molar incorporation of approximately 2-4 biotins per immunoglobulin molecule. 

The following protocol is a suggested method for modifying sulfhydryl-containing proteins with biotin-BMCC. Some optimization of biotinylation levels may have to be done for particular applications. 

The use of discrete PEG spacers in the construction of biotinylation compounds not only increases the water solubility of the modification reagent itself, but significantly increases the hydrophilicity and stability of proteins modified with them. Even when high modification levels... 

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

Dissolve a sulfhydryl-containing protein or other thiol-molecule in a thiol-free buffer within a pH range of 6.5-7.5. The use of 20 mM sodium phosphate, 150 mM NaCl, pH 7.2, works well for this reaction. The concentration of protein should be in the range of l-10mg/ml. Lower concentrations of protein may result in the need to increase the molar excess of biotinylation reagent to obtain an acceptable level of modification. If a... 

Fragmented, biotinylated RNA prepared in this manner was hybridized to the array and the signal developed using streptavidin-R-phycoerytherin. A confocal laser scanner was used for detection. The researchers estimated that they could detect two transcripts per cell based upon labeling efficiency and an estimated 4% mRNA content in total bacterial RNA. Thus, chip detection of labeled transcripts was found to be more sensitive than detection by Northern blot. Specific genes (e.g., basal levels of cinA) undetectable on Northern blots were quantifiable on the microarray. In addition, it was... 

Add 50 xl of the iodoacetyl-LC-biotin solution to each milliliter of the protein solution. Mix well. This level of addition represents a 3.28-fold molar excess of biotinylation reagent over the quantity of protein present if the protein has a molecular weight of 67,000 and possesses one sulfhydryl. Adjustments to the amount of reagent addition may have to be made to be appropriate for other... 

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