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Protein Biotinylation
CHAPTER TEN
Protein Biotinylation Alice Alegria-Schaffer1 Thermo Fisher Scientific, Rockford, IL, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Calculations 5.1 Overview 5.2 Duration 6. Step 2 Protein Biotinylation 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip References
109 110 110 110 111 111 111 111 111 112 113 113 113 113 113 113 114
Abstract Biotin is a naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because biotin is small (244 Da), it can be conjugated to many proteins without altering their biological activities. The biotinylated molecule can be detected in ELISA, dot blot, or Western blot methods (see Western Blotting using Chemiluminescent Substrates) using streptavidin or avidin probes.
1. THEORY Although there are many commercially available biotinylation reagents, the protocol described here uses NHS–PEG4–biotin. This ˚ polyethylene biotinylation reagent contains an NHS-ester group and a 29 A glycol (PEG) biotin linker. NHS esters react with primary amines in slightly alkaline conditions (pH 7.2–8.5) and yield stable amide bonds. The reaction Methods in Enzymology, Volume 536 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420070-8.00010-6
#
2014 Elsevier Inc. All rights reserved.
109
110
Alice Alegria-Schaffer
releases N-hydroxysuccinimide. Proteins typically have many sites for labeling, including the primary amine in the side chain of lysine (K) residues and the N-terminus of each polypeptide (see other application for NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). The hydrophilic (aqueous-soluble) PEG linker imparts water solubility that is transferred to the biotinylated molecule. Consequently, antibodies and other proteins labeled with NHS–PEG4–biotin exhibit less aggregation when stored in solution compared to proteins labeled with reagents having hydrocarbon linkers.
2. EQUIPMENT UV/Vis spectrophotometer Micropipettors Micropipettor tips 1.5-ml polypropylene tubes Device for buffer exchange (e.g., Zeba Spin Desalting column or SlideA-Lyzer dialysis cassette)
3. MATERIALS NHS–PEG4–biotin Bradford reagent Sodium phosphate monobasic (NaH2PO4) Sodium phosphate dibasic heptahydrate (Na2HPO4 7H2O) Sodium chloride (NaCl) HEPES (optional)
3.1. Solutions & buffers Step 1 Phosphate-buffered Saline (PBS)* Component
Final concentration
Stock
Amount
NaH2PO4
100 mM
0.2 M
14 ml
0.2 M
36 ml
Na2HPO4 7H2O NaCl
150 mM
0.88 g
Adjust the pH to 7.2–7.4 (if needed). Add water to 100 ml * Any non-amine-containing buffer at pH 7–9 may be used. Examples include 20 mM HEPES; 100 mM carbonate/bicarbonate; or 50 mM borate
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Protein Biotinylation
4. PROTOCOL 4.1. Preparation Prepare the PBS or other non-amine conjugation buffer. If the protein is in an amine-containing buffer, such as Tris or glycine, perform a buffer exchange (i.e., dialysis or desalting) to effectively remove these products. Determine protein concentration (see Quantification of Protein Concentration using UV absorbance and Coomassie Dyes).
4.2. Duration Preparation
15 min to 3 h
Protocol
45 min to 2 h
See Fig. 10.1 for the flowchart of the complete protocol.
5. STEP 1 CALCULATIONS 5.1. Overview Determine the amount of NHS–PEG4–biotin needed to achieve the appropriate molar excess.
Figure 10.1 Flowchart of the complete protocol, including preparation.
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5.2. Duration 1–5 min 1.1 Determine the desired molar-fold excess of NHS–PEG4–biotin to use. The amount of biotin reagent to use for each reaction depends on the amount and concentration of protein to be labeled. By using the appropriate biotin-to-protein molar ratio, the extent of labeling can be controlled. For dilute protein solutions (e.g., 2 mg ml 1), a greater fold molar excess of biotin is used compared to a concentrated protein solution (e.g., 10 mg ml 1). For example, use a 20-fold molar excess of biotin for a 2 mg ml 1 solution of IgG and use a 12-fold molar excess of biotin for a 10 mg ml 1 solution of IgG. Typically, 3–5 biotin molecules per protein molecule is desirable. Adjust the NHS–PEG4–biotin-to-protein molar ratio to optimize the biotinylation level. 1.2 Calculate the number of millimoles of biotin reagent needed to add to the reaction to give a 20-fold molar excess of biotin (Fig. 10.2). 1.3 Calculate the number of microliters of 20 mM biotin reagent solution (prepared in Step 2.1) needed to add to the reaction (Fig. 10.2).
Figure 10.2 Example calculations to determine how much of a 20 mM solution of biotin to add to 1 ml of a 2 mg ml 1 solution of IgG to give a 20-fold molar excess of biotin.
Protein Biotinylation
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6. STEP 2 PROTEIN BIOTINYLATION 6.1. Overview Proteins are biotinylated with NHS–PEG4–biotin.
6.2. Duration 1.5–3 h 2.1 Immediately before use, add 170 ml of water to 2 mg of NHS–PEG4– biotin to prepare a 20 mM stock solution. 2.2 Add the appropriate volume of the NHS–PEG4–biotin solution (see Calculations section) to the protein solution. 2.3 Incubate reaction on ice for 2 h or at room temperature for 30 min. 2.4 Remove nonreacted NHS–PEG4–biotin by dialysis or gel filtration. See instructions provided with the buffer exchange product. 2.5 Store the biotinylated protein using the same conditions that are optimal for the non-biotinylated protein.
6.3. Tip NHS–PEG4–biotin is moisture-sensitive. To avoid moisture condensation onto the product, the vial must be equilibrated to room temperature before opening.
6.4. Tip Use reconstituted NHS–PEG4–biotin immediately. The NHS–ester moiety readily hydrolyzes and becomes nonreactive; therefore, do not prepare aqueous solutions for storage. Discard any unused reconstituted reagent. It is possible to make a stable concentrated (e.g., 100–200 mM) stock solution by dissolving NHS–PEG4–biotin in pure, moisture-free (‘dry’) DMSO or DMF. With proper handling (i.e., complete exclusion of moisture), the stock could be stable for several months at 20 C.
6.5. Tip The biotinylation level can be determined using the HABA/avidin method. This method is commercially available in a kit format. See Fig. 10.3 for the flowchart of Step 2.
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Figure 10.3 Flowchart of Step 2.
REFERENCES Related Literature Hermanson, G. T. (2008). In Bioconjugate Techniques (pp. 727–730) (2nd ed.). New York: Academic Press. Pierce Biotechnology (2010) Product instructions for EZ-Link® NHS–PEG4–biotin. Document #1299.6.
Referenced Protocols in Methods Navigator Western Blotting using Chemiluminescent Substrates. Labeling a protein with fluorophores using NHS ester derivitization. Quantification of Protein Concentration using UV absorbance and Coomassie Dyes.
Protein Biotinylation Alice Alegria-Schaffer1 Thermo Fisher Scientific, Rockford, IL, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 2. Equipment 3. Materials 3.1 Solutions & buffers 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Calculations 5.1 Overview 5.2 Duration 6. Step 2 Protein Biotinylation 6.1 Overview 6.2 Duration 6.3 Tip 6.4 Tip 6.5 Tip References
109 110 110 110 111 111 111 111 111 112 113 113 113 113 113 113 114
Abstract Biotin is a naturally occurring vitamin that binds with high affinity to avidin and streptavidin proteins. Because biotin is small (244 Da), it can be conjugated to many proteins without altering their biological activities. The biotinylated molecule can be detected in ELISA, dot blot, or Western blot methods (see Western Blotting using Chemiluminescent Substrates) using streptavidin or avidin probes.
1. THEORY Although there are many commercially available biotinylation reagents, the protocol described here uses NHS–PEG4–biotin. This ˚ polyethylene biotinylation reagent contains an NHS-ester group and a 29 A glycol (PEG) biotin linker. NHS esters react with primary amines in slightly alkaline conditions (pH 7.2–8.5) and yield stable amide bonds. The reaction Methods in Enzymology, Volume 536 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420070-8.00010-6
#
2014 Elsevier Inc. All rights reserved.
109
110
Alice Alegria-Schaffer
releases N-hydroxysuccinimide. Proteins typically have many sites for labeling, including the primary amine in the side chain of lysine (K) residues and the N-terminus of each polypeptide (see other application for NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). The hydrophilic (aqueous-soluble) PEG linker imparts water solubility that is transferred to the biotinylated molecule. Consequently, antibodies and other proteins labeled with NHS–PEG4–biotin exhibit less aggregation when stored in solution compared to proteins labeled with reagents having hydrocarbon linkers.
2. EQUIPMENT UV/Vis spectrophotometer Micropipettors Micropipettor tips 1.5-ml polypropylene tubes Device for buffer exchange (e.g., Zeba Spin Desalting column or SlideA-Lyzer dialysis cassette)
3. MATERIALS NHS–PEG4–biotin Bradford reagent Sodium phosphate monobasic (NaH2PO4) Sodium phosphate dibasic heptahydrate (Na2HPO4 7H2O) Sodium chloride (NaCl) HEPES (optional)
3.1. Solutions & buffers Step 1 Phosphate-buffered Saline (PBS)* Component
Final concentration
Stock
Amount
NaH2PO4
100 mM
0.2 M
14 ml
0.2 M
36 ml
Na2HPO4 7H2O NaCl
150 mM
0.88 g
Adjust the pH to 7.2–7.4 (if needed). Add water to 100 ml * Any non-amine-containing buffer at pH 7–9 may be used. Examples include 20 mM HEPES; 100 mM carbonate/bicarbonate; or 50 mM borate
111
Protein Biotinylation
4. PROTOCOL 4.1. Preparation Prepare the PBS or other non-amine conjugation buffer. If the protein is in an amine-containing buffer, such as Tris or glycine, perform a buffer exchange (i.e., dialysis or desalting) to effectively remove these products. Determine protein concentration (see Quantification of Protein Concentration using UV absorbance and Coomassie Dyes).
4.2. Duration Preparation
15 min to 3 h
Protocol
45 min to 2 h
See Fig. 10.1 for the flowchart of the complete protocol.
5. STEP 1 CALCULATIONS 5.1. Overview Determine the amount of NHS–PEG4–biotin needed to achieve the appropriate molar excess.
Figure 10.1 Flowchart of the complete protocol, including preparation.
112
Alice Alegria-Schaffer
5.2. Duration 1–5 min 1.1 Determine the desired molar-fold excess of NHS–PEG4–biotin to use. The amount of biotin reagent to use for each reaction depends on the amount and concentration of protein to be labeled. By using the appropriate biotin-to-protein molar ratio, the extent of labeling can be controlled. For dilute protein solutions (e.g., 2 mg ml 1), a greater fold molar excess of biotin is used compared to a concentrated protein solution (e.g., 10 mg ml 1). For example, use a 20-fold molar excess of biotin for a 2 mg ml 1 solution of IgG and use a 12-fold molar excess of biotin for a 10 mg ml 1 solution of IgG. Typically, 3–5 biotin molecules per protein molecule is desirable. Adjust the NHS–PEG4–biotin-to-protein molar ratio to optimize the biotinylation level. 1.2 Calculate the number of millimoles of biotin reagent needed to add to the reaction to give a 20-fold molar excess of biotin (Fig. 10.2). 1.3 Calculate the number of microliters of 20 mM biotin reagent solution (prepared in Step 2.1) needed to add to the reaction (Fig. 10.2).
Figure 10.2 Example calculations to determine how much of a 20 mM solution of biotin to add to 1 ml of a 2 mg ml 1 solution of IgG to give a 20-fold molar excess of biotin.
Protein Biotinylation
113
6. STEP 2 PROTEIN BIOTINYLATION 6.1. Overview Proteins are biotinylated with NHS–PEG4–biotin.
6.2. Duration 1.5–3 h 2.1 Immediately before use, add 170 ml of water to 2 mg of NHS–PEG4– biotin to prepare a 20 mM stock solution. 2.2 Add the appropriate volume of the NHS–PEG4–biotin solution (see Calculations section) to the protein solution. 2.3 Incubate reaction on ice for 2 h or at room temperature for 30 min. 2.4 Remove nonreacted NHS–PEG4–biotin by dialysis or gel filtration. See instructions provided with the buffer exchange product. 2.5 Store the biotinylated protein using the same conditions that are optimal for the non-biotinylated protein.
6.3. Tip NHS–PEG4–biotin is moisture-sensitive. To avoid moisture condensation onto the product, the vial must be equilibrated to room temperature before opening.
6.4. Tip Use reconstituted NHS–PEG4–biotin immediately. The NHS–ester moiety readily hydrolyzes and becomes nonreactive; therefore, do not prepare aqueous solutions for storage. Discard any unused reconstituted reagent. It is possible to make a stable concentrated (e.g., 100–200 mM) stock solution by dissolving NHS–PEG4–biotin in pure, moisture-free (‘dry’) DMSO or DMF. With proper handling (i.e., complete exclusion of moisture), the stock could be stable for several months at 20 C.
6.5. Tip The biotinylation level can be determined using the HABA/avidin method. This method is commercially available in a kit format. See Fig. 10.3 for the flowchart of Step 2.
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Figure 10.3 Flowchart of Step 2.
REFERENCES Related Literature Hermanson, G. T. (2008). In Bioconjugate Techniques (pp. 727–730) (2nd ed.). New York: Academic Press. Pierce Biotechnology (2010) Product instructions for EZ-Link® NHS–PEG4–biotin. Document #1299.6.
Referenced Protocols in Methods Navigator Western Blotting using Chemiluminescent Substrates. Labeling a protein with fluorophores using NHS ester derivitization. Quantification of Protein Concentration using UV absorbance and Coomassie Dyes.