Protein Protocols Protein Protocols

408 Kruger
7. Horseradish peroxidase substrate mixture:
a. Make up 50 mM acetic acid and adjust to pH 5.0 using NaOH. This buffer is usually
made up as required, but may be stored at 4°C if care is taken to avoid microbial
contamination.
b. Diaminobenzidine stock solution of 1 mg/mL dissolved in acetone. Store in the dark at
–20°C. Caution: Diaminobenzidine is potentially carcinogenic; handle with care.
c. Hydrogen peroxide at a concentration of 30% (v/v). This compound decomposes, even
when stored at 4°C. The precise concentration of the stock solution can be determined
by measuring its absorbance at 240 nm. The molar extinction coefficient for H 2O 2 is
43.6 M –1 cm –1 at this wavelength (see Note 6).
Combine 50 mL of acetate buffer, 2 mL of diaminobenzidine stock solution, and 30 µL
of hydrogen peroxide immediately before use. Mix gently and avoid vigorous shaking
to prevent unwanted oxidation of the substrate. Protect the solution from the light.
8. Protein staining solutions: These are stable at room temperature for several weeks and the
stains may be reused.
a. Amido Black stain (100 mL): 0.1% (w/v) Amido Black in 25% (v/v) propan-2-ol, 10%
(v/v) acetic acid.
b. Amido Black destain (400 mL): 25% (v/v) propan-2-ol, 10% (v/v) acetic acid.
c. Ponceau S stain (100 mL): 0.2% (w/v) Ponceau S, 10% (w/v) acetic acid.
d. Ponceau S destain (400 mL): distilled water.
3. Methods
3.1. Immunodetection of Polypeptides
1. Following SDS-PAGE (see Chapter 11), electroblot the polypeptides from the gel onto
nitrocellulose at 50 V for 3 h using a Bio-Rad Trans-Blot apparatus, or at 100 V for 1 h
using a Bio-Rad Mini Trans-Blot system.
2. After blotting, transfer the nitrocellulose filters individually to plastic trays for the subsequent
incubations. Ensure that the nitrocellulose surface that was closest to the gel is
uppermost. Do not allow the filter to dry out, as this often increases nonspecific binding
and results in heavy, uneven background staining. The nitrocellulose filter should be
handled sparingly to prevent contamination by grease or foreign proteins. Always wear
disposable plastic gloves, and only touch the edges of the filter.
3. If desired, stain the blot for total protein using Ponceau S as described in Subheading
3.3.2. (see Note 7).
4. Rinse the nitrocellulose briefly with 100 mL of PBS. Then incubate the blot at room
temperature with the following solutions, shaking gently (see Note 8).
a. 50 mL of PBS–8% bovine serum albumin for 30 min. This blocks the remaining
protein-binding sites on the nitrocellulose (see Note 9).
b. 50 mL of PBS–8% bovine serum albumin containing 50–500 mL of antiserum for
2–16 h (see Note 10).
c. Wash the nitrocellulose at least 5×, each time using 100 mL of PBS for 15 min, to
remove unbound antibodies.
d. 50 mL of PBS–4% bovine serum albumin containing an appropriate ligand for 2 h (see
Note 11). This is likely to be one of the following:
i. enzyme-conjugated secondary antibody at the manufacturer recommended
dilution (normally between 1:1000 and 1:10,000).
ii. 5 µg of enzyme-conjugated protein A.
iii. 1 µCi 125I-labeled protein A.