- Polymeric microspheres (often supplied at 10% solids)
- Adsorption Buffer (low ionic strength buffer of pH at or near pI of protein)
- Purified ligand
- Storage buffer (adsorption buffer with 0.01-0.1% blocking molecule added)
- Dilute the microspheres to 1% solids (10 mg/ml) with adsorption buffer.
Note: Although surfactants or detergents, in which microspheres are normally shipped, may sometimes interfere with binding, it is often not necessary to clean the microspheres prior to use.
If cleaning is desired, this can be done by techniques like centrifugation, dialysis, or ion exchange as described in our
- Dissolve appropriate amount of purified ligand in adsorption buffer.
- Add the microsphere suspension to the appropriate volume of dissolved protein, and mix gently for 1-2 hours.
Note: By adding microspheres to protein, rather than protein to microspheres, efficiency is maximized and even distribution of adsorption is more likely.
- Incubate suspension overnight at 4°C, with constant mixing.
Note: Although the vast majority of ligand adsorption occurs very rapidly, the extended incubation seems to aid in achieving correct orientation by allowing a equilibrium to be reached.
Other options are to incubate at room temperature for 1-2 hours, or at 37°C for 15-30 minutes (in cases where the ligand will not be adversely affected by elevated temperatures).
- Centrifuge, remove supernatant*, and resuspend microspheres pellet in storage buffer to desired storage concentration (often 10 mg/ml). A separate blocking step may be added here if necessary.
* Supernatant can be saved to determine the amount of free protein, from which the amount of adsorbed protein can be indirectly quantified.
A common assay to determine the amount of free protein in solution is the BCA assay (Pierce Chemical Company).
A more crude measurement can be made by measuring the A 280 of the supernatant on a spectrophotometer.
Absorbance at 280 nm: A280
- Spectrophotometer (equipped for UV reading)
- Matched Quartz Cuvettes
- Pasteur pipettes and pipette bulbs for solution transfer
- Adsorption Buffer (for blank)
- Supernatant (from Procedure a)
Single Beam Spectrophotometer
- With no cuvette present in instrument, set A 280 to zero.
- With adsorption buffer in cuvette, read A 280, then reset to zero (this step determines whether the adsorption buffer has a significant absorbance.)
- Remove buffer and add supernatant to cuvette, then record absorbance.
Dual Beam Spectrophotometer
- With matched, empty cuvettes in machine, set instrument zero.
- Add adsorption buffer to sample cuvette, leave reference cuvette empty. Record absorbance (this step determines whether the buffer has a significant absorbance.)
- Remove buffer from sample cuvette. Add supernatant and adsorption buffer to sample and reference cuvettes, respectively, then record absorbance.
- It is a common laboratory shortcut (although a very imprecise one) to assume that an absorbance of 1.0 in a 1 cm cuvette roughly approximates 1 mg/ml of protein.
For comparison, measured A 280 values of a sampling of proteins at 1 mg/ml follow: Bovine Serum Albumin 0.70; Ovalbumin 0.79; gamma-Globulin 1.38; Trypsin 1.60; Chymotrypsin 2.02; a-Amylase 2.42
- If absorbance is off scale, the sample can be diluted with buffer and the assay repeated. Alternatively, a cuvette with a shorter path length may be used.
- Glass or plastic cuvettes absorb light in the UV range and should not be use for this assay.
Covalent Coupling to Non-functionalized Polymeric Microspheres
Although adsorption of hydrophobic ligands to polymeric microspheres is advantageous in many situations, there are times when the hydrophobic attractive forces may not be strong enough to resist the incubation and wash steps included in many assay procedures.
In other cases, the antibody in question might not be able to be adsorbed and still retain its immunoreactivity. One answer to such situations is to modify the surface of the microspheres so that covalent coupling becomes an option. Following are approaches that can be taken for such modification.
- A practical and reproducible coating method for plain polystyrene involves the adsorption of dial and coupling of the required ligand.
The poly phe-lys fulfills two important requisites.
First, the strong hydrophobic interactions between the phenylalanine residues and the polystyrene surface create an essentially irreversible adsorption.
Secondly, the introduction of 'reactive' amino groups offers a means of co-valent attachment via standardized chemistries.
- It is possible to directly derivatize, and covalently couple ligands to, non-functionalized polystyrene microspheres.
Many other surface functionalized microspheres are available for easy covalent coupling.
Following are some basic recipes for buffers commonly used in adsorption protocols. Generally, maximal adsorption occurs at or near the pI of the protein, and so the choice of buffer should be made accordingly.
Additionally, many researchers have reported that the addition of NaCl to the coupling buffer, in physiological concentrations of about 0.15 M, increases adsorption efficiency.
This information is intended only as a general guideline. Feel free to substitute buffers and/or adjust concentrations as your application demands.
Phosphate Buffered Saline (PBS); pH 7.4
- Potassium Phosphate dibasic: 1.82 g/l (MW 174.2)
- Sodium Phosphate monobasic: 0.22 g/l (MW 120.0)
- Sodium Chloride: 8.76 g/l (MW 58.4)
Bring to final volume of 1 L using DI water. Adjust pH to 7.4 using either 1N HCl or 1N NaOH.
Borate Buffer, pH 8.5
- Boric Acid, H3BO3: 12.4 g/l(MW 61.8)
- Sodium Tetraborate: 19.1 g/l(MW 381.4)
Add 50 ml of (1) to 14.5 ml of (2).
Bring to final volume of 200 ml using deionised water. Adjust pH to 8.5 with NaOH (3M).
Acetate Buffer; pH range 3.6 to 5.6
- 0.1 M Acetic acid (5.8 ml made to 1000 ml)
- 0.1 M Sodium acetate; 8.2 g/l (anhydrous, MW 82.0)
Mix acetic acid and sodium acetate solutions in the proportions indicated and adjust the final volume to 100 ml with deionised water. Adjust the final pH using HCl (1 N) or NaOH (.1 N).
Citrate-Phosphate Buffer; pH range 2.6 to 7.0
- 0.1 M Citric acid; 19.2 g/l (MW 192.1)
- 0.2 M Dibasic sodium phosphate; 35.6 g/l (dihydrate; MW 178.0)
Mix citric acid and sodium phosphate solutions in the proportions indicated and adjust the final volume to 100 ml with deionized water. Adjust the final pH using 1 N HCl or 1 N NaOH.
Carbonate-Bicarbonate Buffer; pH range 9.2 to 10.4
- 0.1 M Sodium carbonate (anhydrous), 10.6 g/l (MW 106.0)
- 0.1 M Sodium bicarbonate, 8.4 g/l (MW 84.0)
Mix sodium carbonate and sodium bicarbonate solutions in the proportions indicated and adjust the final volume to 200 ml with DI water. Adjust the final pH using 1 N HCl or 1 N NaOH.
Note: Small concentrations of anti-microbial agents (0.05-0.1% w/v) such as sodium azide or merthiolate are often added to the storage buffer, especially for long-term storage.
Blockers can be added to the storage buffer in varying amounts, a standard concentration being 0.05 %(w/v).
Using a substance dissolved in the storage buffer that will block the exposed hydrophobic surfaces of the polymeric microspheres will reduce non-specific binding and self-aggregaton of the microspheres.
A separate incubation in a higher concentration of blocker (up to 0.1%) is also recommended before storage, in order to saturate the exposed hydrophobic surfaces of the microspheres.
Some commonly used blockers are as follows:
- BSA (Bovine Serum Albumin): Often used alone, but can be combined with other blockers, most commonly surfactants.
- Casein: A milk-based protein, containing indigenous biotin, which should be avoided when working with systems involving biotin to prevent interference.
- Pepticase(Casein Enzymatic Hydrolysate): an enzymatic derivative of casein, should also be avoided when working with systems involving biotin.
- Non-Ionic Surfactants: Tween 20 and Triton X-100 are typical. When used in combination with another blocker, a common ratio is 1% Blocker: 0.05% Surfactant.
- "Irrelevant" IgG: Often used when conjugating a specific IgG to microspheres. For example, if coupling mouse IgG, rabbit (or any non-cross reacting IgG)can be adsorbed as a blocker.
- FSG (Fish Skin Gelatin): Pure gelatin or gelatin hydrolysate can also be used.
- Polyethylene Glycol: A very versatile blocker, available in a number of sizes, configurations, and charges.
- Sera: Non-cross-reacting serums, such as horse or fish se-rum, are very inert in terms of cross- reactivity with various types of antibodies.
- Commercial Blockers: Many companies offer preparations which are a composite of 2 or more single blocking substances of various molecular weights, and which can be used effec-tively over a wide range of conditions.
These go under various trade names, and most chemical vendors will offer a variety of these.
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