Copper Iodide Staining of Proteins on Solid Phases

Abstract

Copper iodide staining and silver enhancement are recent implementations of copper-based protein assays, and are designed to quantitate proteins adsorbed to solid surfaces, such as nitrocellulose, nylon, polyvinylenedifluoride, and polystyrene (1–5). The binding of cupric ions to the backbone of proteins under alkaline conditions and their consequent reduction to the cuprous state are the basis of popular assays of protein in solution including the biuret, Lowry, and bicinchoninic acid methods (see Chapters 2 and 3). In the case of copper iodide staining, it is thought that the protein binds copper iodide under highly alkaline conditions. Sensitivity, speed, reversibility, low cost, and lack of known interfering substances (including nucleic acid) are among the virtues of this protein assay (4–6). One interesting use of copper iodide staining is the quantification of protein adsorbed to microtiter plates (5). This information is particularly useful for quantitative ELISA and protein binding when radioactive measurements are inconvenient or undesirable (e.g., chemical modification affects the properties of the protein of interest). The precision of the determination of protein adsorbed to the microtiter plate by copper iodide staining is typically about 10–15%. The high sensitivity of copper iodide staining (about 50 pg/mm²) may be increased an additional 10-fold by a silver-enhancement procedure, allowing the detection of protein down to about 5 pg/mm² (6). Protein concentrations may be estimated from copper iodide staining from very dilute protein solutions or when only small amounts of a precious protein are available.

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1 Introduction

Copper iodide staining and silver enhancement are recent implementations of copper-based protein assays, and are designed to quantitate proteins adsorbed to solid surfaces, such as nitrocellulose, nylon, polyvinylenedifluoride, and polystyrene (1–5). The binding of cupric ions to the backbone of proteins under alkaline conditions and their consequent reduction to the cuprous state are the basis of popular assays of protein in solution including the biuret, Lowry, and bicinchoninic acid methods (see Chapters 2 and 3). In the case of copper iodide staining, it is thought that the protein binds copper iodide under highly alkaline conditions. Sensitivity, speed, reversibility, low cost, and lack of known interfering substances (including nucleic acid) are among the virtues of this protein assay (4–6). One interesting use of copper iodide staining is the quantification of protein adsorbed to microtiter plates (5). This information is particularly useful for quantitative ELISA and protein binding when radioactive measurements are inconvenient or undesirable (e.g., chemical modification affects the properties of the protein of interest). The precision of the determination of protein adsorbed to the microtiter plate by copper iodide staining is typically about 10–15%. The high sensitivity of copper iodide staining (about 50 pg/mm²) may be increased an additional 10-fold by a silver-enhancement procedure, allowing the detection of protein down to about 5 pg/mm² (6). Protein concentrations may be estimated from copper iodide staining from very dilute protein solutions or when only small amounts of a precious protein are available.

2 Materials

2.1 Copper Iodide Staining

• 1. Prepare the copper iodide staining reagent by mixing CuSO₄·5H₂O (12 g), KI (20 g), and potassium sodium tartrate (36 g) with 80 mL of distilled water in a glass beaker (see Note 1). As the slurry is vigorously stirred, solid NaOH (10 g) is slowly added. The suspension becomes warmer and changes color from brown to green to dark blue. After the NaOH is completely dissolved, the beaker is allowed to cool at room temperature for 30 min without stirring to allow the brownish-red precipitate to settle. Then 70 mL of solution are aspirated from the top to leave approx 50 mL of reagent with precipitate. The reagent is stable and may be stored in a sealed bottle at room temperature for at least 1 mo or at 4°C for at least 1 yr (see Note 2).

• 2. Prepare the copper iodide stain-remover solution with 0.19 g Na₄EDTA·H₂O, 0.28 g NaH₂PO₄·H₂O, and 2.14 g Na₂HPO₄·7H₂O in 100 mL deionized water.

• 3. Nitrocellulose (e.g., BA85, Schleicher & Schuell, Keene, NH), polyvinylenedifluoride (e.g., PVDF, Millipore Corporation, Bedford, MA), or nylon blotting paper (e.g., Zeta probe, Bio-Rad Laboratories, Hercules, CA; (see Note 3).

2.2 Silver-Enhanced Copper Staining (SECS)

• 1. Prepare the silver-enhancing reagent just prior to use by dissolving 0.1 g AgNO₃, 0.1 g NH₄NO₃, and 7 µL of 5% (v/v in water) β-mercaptoethanol in 100 mL distilled water. After the other components are dissolved and immediately before use, add 2.5 g Na₂CO₃.

• 2. Prepare the SECS stain remover by dissolving 33.2 g KI in 100 mL distilled water (final concentration, 2M KI).

2.3 Copper Iodide Microtiter Plate Assay

• 1. Polystyrene 96-well microtiter plates (e.g., Nunc Immuno-Plate, Denmark; Titertek 76-381-04, McLean, VA; or Immulon 1 and 2, Dynatech Laboratories, Chantilly, VA).

• 2. Nitrocellulose membranes (e.g., BA85, Schleicher & Schuell, Keene, NH).

• 3. Household 3-in-one lubricating oil (Boyle-Midway, Inc., New York).

• 4. A standard single-hole puncher (6-mm diameter).

• 5. A densitometer is required, such as a flatbed scanner or video camera and framegrabber with image analysis software (see Note 4).

3 Methods

3.1 Copper Iodide Staining

• 1. Stir the copper iodide staining reagent vigorously at room temperature immediately prior to use. The reagent should be a fine slurry.

• 2. Rock the copper iodide staining reagent over the dried protein blot (or Western blot) for at least 2 min (but not more than 5 min; see Note 5) as the reddish-brown bands appear on the blot.

• 3. Gently dip the stained blot up and down in three beakers of deionized water and then allow the blot to dry (see Note 6).

• 4. The stained blot then may be quantified by densitometry (see Note 7) and photographed for documentation.

• 5. The staining pattern is stable at room temperature for at least 1 yr.

• 6. If greater sensitivity is desired, the blot may be used directly in the silver-enhanced copper staining (SECS) procedure (see Section 3.2.).

• 7. If subsequent immunostaining on the same blot is required, destain for 15 min with gentle agitation in the copper iodide stain remover prior to immunostaining.

3.2 SECS

• 1. Rock a nitrocellulose protein blot stained with copper iodide (see Section 3.1.) for 5 min in freshly prepared silver-enhancing reagent until the bands become dark black.

• 2. Dip the blot in a 1 L beaker of deionized water, and store in the dark to dry (to prevent background development).

• 3. The blot then may be quantified by densitometry and photographed for documentation.

• 4. If subsequent immunostaining on the same blot is required, destain for 30 min with gentle agitation in the SECS stain remover prior to immunostaining (see Note 8).

Fig. 1.

Schematic diagram of the assembly of a microtiter plate for the copper iodide staining assay. Avoid wells marked with “X” because of possible distortions from edge effects.

3.3 Copper Iodide Microtiter Plate Assay

• 1. Adsorb the protein of interest to duplicate microtiter plates, and note the volume (V) that was used to adsorb the protein in each well (see Note 9). One of the microtiter plates is for copper iodide staining, and the other is for quantitative ELISA or binding experiments. The microtiter plate for copper iodide staining contains protein adsorbed to only a few of the wells (e.g., 4–16 wells; (see Fig. 1 and Note 10).

• 2. Create a standard curve by dot blotting approx 5–100 ng of the protein of interest/5 µL drop onto nitrocellulose paper, and allow to air-dry. Blanks are dotted with equal volumes of buffers.

• 3. Stain both the nitrocellulose paper and the microtiter plate with adsorbed protein by the copper iodide staining procedure (see Section 3.1.).

• 4. Use the hole puncher to excise stained dots from the nitrocellulose membrane, and place them stained side down into blank wells on the microtiter plate (see Note 11).

• 5. Use the hole puncher to excise blank nitrocellulose circles, and place them in microtiter plate wells containing copper iodide-stained protein and also in some blank wells to determine the background density (see Fig. 1).

• 6. For transmittance densitometry, first add 5 µL/well of household three-in-one oil to make the nitrocellulose translucent, thus reducing the background. For reflectance densitometry, the bottom of the microtiter plate may be scanned directly (the stained sides of the nitrocellulose must all be face down in the wells).

• 7. Measure the mean optical density and total area of the known amounts of stained protein (adsorbed to nitrocellulose) and construct a standard curve of mean optical density vs ng/mm² of protein (see Note 12).

• 8. Measure the mean optical density of stained sample protein (adsorbed to microtiter plate), and compare to the standard curve to determine the concentration of protein (in ng/mm²) on the microtiter plate well.

• 9. Calculate the total area of the stained sample protein (adsorbed to microtiter plate) on the microtiter plate well from the equation:

  

  in which r (in mm) is the radius of the cylindrical flat-bottom well, and V (in µL) is the volume that was used to adsorb protein to the well. The total area (typically 94.7 mm² for a 100-µL volume applied to a plate with a 3.25-mm well radius) multiplied by the surface density (in ng/mm²) of the sample protein yields the amount of protein (in ng) adsorbed to each microtiter plate well.

4 Notes

• 1. Proportions of sodium and potassium ions in the copper iodide staining reagent are important. Thus, potassium sodium tartrate (sodium potassium tartrate) should not be substituted with, for instance, sodium tartrate.

• 2. The copper iodide staining reagent can generally be reused two to three times, but will eventually become less sensitive.

• 3. Copper iodide staining reagent stains proteins adsorbed on most solid-phase adsorbents, including nitrocellulose, nylon, polyvinylenedifluoride, and polystyrene. However, only nitrocellulose and polystyrene have been tested so far for quantitation.

• 4. Microtiter plate readers are to be avoided for quantitative measurements, because they are usually not sensitive enough (copper iodide staining yields OD ≤ 0.1) and do not sample a large enough area of the stained surface to detect any nonuniformity in the staining density.

• 5. Exceeding a staining time of 5 min can both damage nitrocellulose membranes and lead to solubilization of adsorbed protein. A staining time of 2 min is optimal.

• 6. Washing of microtiter plates should be handled gently by dipping in beakers of deionized water. Vigorous washing procedures often lead to nonuniform protein distribution and consequent uneven staining of microtiter plates.

• 7. An example of a low-cost densitometer is a desktop flatbed scanner, color Apple Macintosh computer, and NIH Image software (public domain, by Wayne Rasband; further details are available by personal communication).

• 8. SECS may be removed by concentrations of KI that are <2M, but will require longer incubations (e.g., 90 min for 0.5M KI).

• 9. The wells on the edge of microtiter plates should be avoided for quantitative measurements because they tend to yield less accurate numbers.

• 10. Nitrocellulose quantitatively binds most proteins that are dot-blotted onto it, and retains them well throughout copper iodide staining.

• 11. If there is a problem with static repulsion, the household three-in-one oil (see Section 3.3., steps 5 and 6) may be applied to the microtiter plate well to release the static charge before placing nitrocellulose circles in the wells (provided that transmission densitometry will be used).

• 12. Quantitative measurements of copper iodide staining should be done at least in triplicate because of the relatively high (10–15%) standard deviation of the results.