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Biotinylation of Antibodies in Phage Display
Contributor:
The Laboratory of George P. Smith at the University of Missouri
URL: G. P. Smith Lab Homepage
Overview
Biotinylated antibodies are used for phage capture assays to isolate bacteriophage particles displaying proteins of interest (see Protocol ID#2179, Protocol ID#2162, and Protocol ID#2167). Biotin is linked to the exposed amino groups of an antibody or other protein by acylation with a biotinylating reagent (NHS-LC-Biotin, Pierce). Once the biotins are attached, the modified antibodies are bound to streptavidin on a solid substrate.
Procedure
1. Pipette 10 to 50 μg of IgG antibody (see Hint #2) into a siliconized microcentrifuge tube (see Hint #3).
2. Add 4.4 μl of 1 M NaHCO3 to the IgG.
3. Add sufficient ddH2O to bring the total volume to 39 μl (see Hint #4).
4. Dissolve approximately 1 mg of the Biotinylating Agent into 2 ml of Sodium Acetate Buffer (see Hint #5).
5. Immediately (see Hint #6) add 5 μl of the solution prepared in Step #4 to the antibody solution prepared in Step #3.
6. Allow the reaction to proceed for 2 hr at room temperature (see Hint #7, #8).
7. Add 500 μl of Ethanolamine Buffer.
8. Allow the reaction to continue for 2 hr at room temperature (see Hint #9).
9. Add 20 μl of Dialyzed BSA (see Hint #10).
10. Add 1 ml of TBS (see Hint #11).
11. Pipette sample onto a Centricon 30 ultrafilter (see Hint #12, #13). Concentrate samples following manufacturer's instructions.
12. Wash the retained product (retentate) three times with 2 ml of TBS to remove all the unconjugated biotin.
13. Wash the retentate once with TBS/0.02% NaN3.
14. Collect the retentate into the conical cup by back-centrifugation as described in the manufacturer's instructions for Centricon.
15. Store the retentate in a 500 μl tube at 4°C.
15. Measure the volume with a micropipetter (see Hint #14); typically we obtain 50 to 80 μl. For long term storage, add an equivalent volume of ultrapure glycerol and store the mixture at -20°C (see Hint #15).
Solutions
Biotinylating Agent
NHS-LC-Biotin (Pierce)
1 M NaHCO3
Filter sterilize
TBS/0.02% NaN3
TBS/0.02% NaN3
TBS/0.02% NaN3
Prepare in 1X TBS
0.02% NaN3
TBS (1X)
50 mM Tris HCl, pH 7.5
Store at room temperature
Autoclave if desired
150 mM NaCl
Dialyzed BSA
50 mg/ml Biotin-free Bovine Serum Albumin (BSA) from Sigma
Store at -20°C
Filter sterilize
Prepare in ddH2O
Ethanolamine Buffer
1 M Ethanolamine (CAUTION! See Hint #1)
Adjust the pH to 9 using HCl
Filter sterilize and store at 4°C
Sodium Acetate Buffer
Adjust the pH to 6
2 M Sodium Acetate
BioReagents and Chemicals
Sodium Bicarbonate
Sodium Azide
Tris HCl
Sodium Chloride
NHS-LC-Biotin
Antibody, IgG
Bovine Serum Albumin
Ethanolamine
Sodium Acetate
Hydrochloric Acid
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. Most water-soluble target receptors (ligates) may be substituted for IgG.
3. Larger amounts of protein may be biotinylated. The volumes of the other components should be increased to keep the concentrations of the components similar to those recommended.
4. The protein solution should contain no primary or secondary amines or other groups that might react with the reagents, other than those on the protein itself. The final pH should be between 7 and 9.
5. The concentration is approximately 0.9 mM
6. In aqueous buffer, hydrolysis of the NHS ester competes with the desired biotinylation reaction. Although the hydrolysis is slow at pH 6, (even in the Sodium Bicarbonate buffer) there should be no delay after dissolving the solid.
7. The final concentration of the biotinylating reagent is approximately 100 μM.
8. The contributors of the protocol have found (in a separate large scale experiment using IgG at a final concentration of 1 mg/ml [6.7 μM-a 15-fold molar deficit relative to the biotinylating agent]) that each antibody molecule incorporates approximately 6 biotin moieties. Such antibodies are effective in biopanning. Since the biotinylation reaction is probably limited by the competing hydrolysis reaction, it is likely to be the concentration of the biotinylating agent (not its molar ratio to the antibody) that determines the degree of biotinylation. The contributors therefore expect the same degree of biotinylation (approximately 6 biotins per molecule) even if the IgG concentration is low (i.e., 230 μg/ml).
9. The ethanolamine reacts with any unreacted biotinylating reagent, preventing biotinylation of the carrier BSA that is to be added in the next step.
10. When an amount greater than or equal to1 mg is used, the carrier BSA can be omitted. This allows the final yield of biotinylated protein to be quantified spectrophotometrically.
11. The subsequent steps may be carried out in non-siliconized labware since the BSA acts as a carrier protein.
12. The Centricon 30 (Amicon) molecular weight cutoff is 30 KDa. Use the appropriate Centricon 30 for your ligate. Process the Centricon according to the manufacturer's instructions.
13. Centricon ultrafilters in this procedure have a capacity of 1 mg. If larger amounts of protein are biotinylated, the contributors suggest the use of dialysis or other larger ultrafilter (for example, Ultrafree 15 from Millipore) to remove unreacted biotin and concentrate the conjugate.
14. The contributors calculate the concentration of biotinylated protein assuming no losses.
15. Some proteins other than antibodies-particularly low-molecular-weight proteins-may be inactivated by high-level biotinylation. If you are uncertain, it may be advisable to biotinylate to a lesser extent. For example, we biotinylated ribonuclease S-protein at a final concentration of 100 μM with biotinylating reagent at a final concentration of 176 μM. At this molar ratio, a substantial fraction of the S-protein molecules are expected to be conjugated to a single biotin. At the same time, a substantial fraction undoubtedly remains unconjugated in these circumstances. In "one-step" affinity selection (see Protocol ID#2179), unconjugated ligate should not interfere as long as the streptavidin-coated dish or well is charged with an excess of biotinylated protein. In "two-step" affinity selection, reaction of target phage with unconjugated ligate (making a complex that cannot be subsequently "captured" on a streptavidin-coated dish or well) competes against reaction with conjugated ligate (making a complex that can be captured); if target phage are limiting, this side-reaction can reduce yield. In most cases, however, this reduction in yield is not severe enough to interfere noticeably with affinity selection.
