Protein Protocols Protein Protocols

548 Judd
easy to use and reliable, but can be toxic, requiring careful handling (see Note 7). Several
practical cleavage reagents are presented in Table 1.
Volatile buffers must be used when using 2-D TLE-TLC peptide separation, since
this system is negatively affected by salts. For formate and CNBr cleavages, the acid is
diluted in H 2O to 88 or 70%, respectively. BNPS-skatole works well in 50% glacial
acetic acid–50% H 2O. Ammonium bicarbonate (50 mM), adjusted to the appropriate
pH with sodium hydroxide, is excellent for enzymes requiring weak base environments
(trypsin, α-chymotrypsin, thermolysin, V8 protease). The acid peptidase, Pepsin A, is
active in H 2O adjusted to pH 3.0 with glacial acetic acid (see also Chapter 76).
3.3.1. Protein on NCP Strip
1 Put the NCP strip containing the radiolabeled protein in a 1.5-mL microfuge tube, and
measure the radioemission using a γ-radiation detector.
2. Add 90 µL of the appropriate buffer and 10 µL of chemical or enzymatic cleavage reagent
in buffer (1 mg/mL) to the NCP strip.
3. Incubate with shaking at 37°C for 4 h (for enzymes) or at room temperature for 24–48 h in
dark under nitrogen (for chemical reagents).
4. Aspirate the peptide-containing supernatant, and count the NCP strip and supernatant.
Enzymes should release 60–70% of counts in the slice into the supernatant; CNBr should
release >80%, BNPS-skatole rarely releases more than 50% (see Notes 8 and 9).
5. Completely dry the supernatant in a Speed-Vac, and wash the sample at least four times
by adding 50 µL of H2O, vortexing, and redrying in a Speed-Vac. Alternate drying systems
will work.
6. The sample is now ready for peptide separation (see Note 10).
3.3.2. Protein in Gel Slice
1. Put the dry gel slice containing the radiolabeled protein in a 1.5-mL microfuge tube and
measure the radioemission using a γ-radiation detector.
2. Add 10 µL of cleavage reagent in buffer (1 mg/mL) directly to the dry gel slice. Allow
slice to absorb cleavage reagent, and then add 90 µL of appropriate buffer.
3. Continue as from step 3, Subheading 3.3.1. Release of peptides into the supernatant will
be less efficient than with the NCP strip.
4. The sample is now ready for peptide separation (see Note 10).
3.3.3. Lyophilized/SolubleProteins
1. Rehydrate the lyophilized radiolabeled proteins in the appropriate buffer at 1 mg/mL (less
concentrated samples can be used successfully).
2. Add up to 25 µL of the appropriate cleavage reagent (1 mg/mL) to 25 µL of suspended
protein.
3. Continue as from step 3, Subheading 3.3.1., except there is no strip to count.
4. The sample is now ready for peptide separation. Be aware that the sample will usually
contain uncleared protein along with the peptide fragments (see Note 10).
3.4. 2-D TLE-TLC Peptide Separation
It is strongly recommended that iodinated samples be used in the 2-D TLE-TLC
system. The technique described is precise enough that peptide maps can be overlaid to
facilitate comparisons. Flat-bed electrophoresis can be used, but systems that cool by
immersion of the thin-layer sheet in an inert coolant, such as “varsol” (such as the
Savant TLE 20 electrophoresis chamber or equivalent), yield superior results. Cooling