Protein Protocols Protein Protocols

546 Judd
4. Excise the protein band from the NCP (a 1 × 5-mm band is more than ample), and place
the excised strip in a 1.5-mL microfuge tube. Wash with H 2O until no stain is released into
the supernatant. The protein is now ready for labeling and cleavage (see Note 4).
3.1.1.2. DOUBLE SDS-PAGE SEPARATION
1. Separate the samples in individual lanes of an SDS-PAGE gel or in “preparative”
SDS-PAGE gels. Fix, stain with CBB, and then destain (see Chapter 11).
2. Excise the protein bands of interest. Soak the bands in 50% ethanol–50% stacking buffer
(1 M Tris-HCl, pH 6.8) for 30 min to shrink the gel strip to facilitate loading onto a second
SDS-PAGE gel.
3. Push the excised band into contact with the stacking gel of a second SDS-PAGE gel of a
different acrylamide concentration (generally use high concentration in the first gel and
lower concentration in the second gel).
4. Separate proteins in a second gel (CBB runs just behind the dye front). Stain or electroblot
the proteins as in Subheading 3.1.1. The protein is now ready for labeling and cleavage.
3.2. Protein Labeling
Proteins can be intrinsically labeled by growing organisms in the presence of a uniform
mixture of 14C-amino acids (5), but this is quite expensive. Intrinsic labeling with
individual amino acids, such as 35S-Met or 35S-Cys, will not work, since many peptide
fragments will not be labeled. Iodination with 125I is inexpensive and reproducible.
Iodinated peptides are readily visualized by autoradiography (1,3,4). Comparative
cleavages of a 40,000-Dalton protein intrinsically labeled with 14C-amino acids extrinsically
labeled with 125I showed that 61 of 66 α-chymotryptic peptides were labeled
with 125I, whereas all 22 Staphylococcus aureus V8 protease-generated peptides were
labeled with 125I, demonstrating the effectiveness of radioiodination (10). This demonstrates
that tyrosine (Tyr) is not the only amino acid labeled using this procedure.
Iodination mediated by chloramine-T (CT) (11) produces extremely high specific
activities, but the procedure requires an extra step to remove the CT and can cleave
some proteins at tryptophan residues (12). This can be beneficial since it is specific and
increases the number of peptides, thus increasing the sensitivity of the procedure (see
ref. 13) for peptide maps of CT- vs Iodogen-labeled proteins). Unfortunately, small
peptides generated by CT cleavage, followed by a second enzymatic or chemical cleavage,
can be lost during the removal of the CT and unbound 125I. The 1,3,4,6-tetrachloro-3α,6α-glycouril (Iodogen) (13) procedure, where the oxidizing
agent is bound to the reaction vessel, does not damage the protein and produces
high specific activities. Aspiration of the reaction mixture stops the iodination and
separates the protein from the oxidant in a single step. For these reasons, Iodogenmediated
labeling is the preferred method for radioiodination. (Radioemission of 125I will be expressed as counts per minute [cpm]. This assumes a detector efficiency of
70%. If detector efficiency varies, multiply the cpm presented here by 1.43 to determine
decays per minute (dpm), and then multiply the dpm by the efficiency of your
detector.)
3.2.1. NCP Strip (Preferred Method)
1. Put the protein-containing NCP strip in an Iodogen-coated (10 µg) microfuge tube.
2. Add 50–100 µL PBS, pH 7.4 (any dilute, neutral buffer should work) and 50–100 µCi 125I (as NaI, carrier-free, 25 µCi/µL) (see Notes 1 and 2).