Amplified Fragment-Length Polymorphism and Protein Profiling for Identification
3.3. Protein Profile Analysis
3.3.1. Preparation of Whole-Cell Lysates
1. Colonies grown on the blood agar plate are harvested by suspending the colonies in several milliliters of PBS (100 mg cells [wet weight] in approx 20 mL of PBS).
2. Add PBS to achieve a final volume of 30 mL.
3. Centrifuge 20 min at 10,000g, discard the supernatant, and wash cells by resus- pending them in 30 mL of PBS. Harvest by centrifuging for 10 min at 10,000g.
4. Repeat washing and centrifuging at least one more time.
5. After the final centrifugation step, discard the supernatant and dissolve 50 mg of the pellet (wet weight) in 0.9 mL of solvent. Sample preparation can be per- formed in 1.5-mL centrifuge tubes. Thoroughly mix the suspension.
6. Boil the pellet/solvent solution for 10 min. Finally, centrifuge for 10 min at 7000g at 4°C to sediment large fragments and unlysed cells.
7. If not used immediately, store the samples at –20°C for short periods or at –80°C for prolonged periods. If stored, boil pellets before electrophoresis.
3.3.2. SDS-PAGE of Whole-Cell Proteins
1. Assemble clean glass plates with spacers in a cassette (see Note 7).
2. Add 20 mL of separation buffer, 32 mL of acrylamide solution, 0.8 mL of 10%
SDS, and 26.8 mL of water. After mixing add 40 µL of TEMED and 0.28 mL of 10% APS. Mix and pour the solution immediately between the plates. Overlay with 2 mL of isobutanol.
3. After 1 h, discard the isobutanol and fill with separation gel buffer. Cover with parafilm and allow the gel to polymerize for 16 to 24 h.
4. Pour off the liquid layer and rinse the gel surface with water.
5. Mix 5 mL of stacking buffer, 3.4 mL of acrylamide solution, 0.2 mL of SDS, and 11.3 mL of water. Add 25 µL of TEMED and 0.1 mL of 10% APS. Stir thor- oughly and pour the solution between the plates. Insert a Teflon comb and re- move airbubbles. Allow the gel to polymerize for 30 min.
6. Mark the position of slots and apply the protein samples in each slot with a microsyringe. Apply the size standard in the wells on both sides of the samples.
7. Run the gel overnight at constant current (6 mA per gel) and temperature.
Fig. 3. Dendrogram derived from unweighted pair group method using average linkage (UPGMA) cluster analysis of amplified fragment-length polymorphism pro- files (50–500 bp) of C. lari. The scale bar indicates levels of linkage between patterns.
The roman numerals indicate the distinct clusters to which the strains belong. The asterisk indicates a highly diverse AFLP pattern that clusters separately. (From ref. 19 with permission from ASM.)
3.3.3 Gel Fixation and Staining
1. Add the gel immediately for fixation in 3% trichloroacetic acid and shake gently for 30 min.
2. Pour of the solution and add staining solution. Shake gently for 1 h.
3. Destain the gel in a solution containing 25% (v/v) methanol and 10% (v/v) acetic acid.
4. Store the gel in a closed container or dry using a gel dryer system.
3.3.4. Computer Analysis of Protein Profiles
1. Take a picture (high resolution) of the gel using a charge-coupled device camera from the laboratory.
2. Digitize the photographs using a flat bed scanner and store as TIFF files.
3. Import the TIFF files in the GelCompar or BioNumerics software. The gels are normalized using the size standard lanes on both sides of the gel.
4. Only the band sizes between 36 kDA and 20.1 kDa are included in the pearson coefficient for similarity calculation and UPGMA cluster analysis (see Note 8).
4. Notes
1. Several enzymes have been used, including EcoRI, PstI, HindIII, and ApaI, com- bined with either MseI or TaqI. For the majority of bacterial genomes, a combi- nation of EcoRI and MseI appears to be the most suitable for AFLP analysis, although not for C. jejuni (because of the fact that EcoRI digestion is inhibited in this species [24]).
2. Compared with a single selective PCR, a preselective AFLP before a selective PCR results in less variation in peak intensities. Also, the stringent PCR annealing temperature yields high reproducibility in the AFLP analysis.
3. Other gel or capillary DNA sequencers and several fluorescently labeled dyes and size markers are available.
4. Acrylamide is neurotoxic and should be handled with care. Store dark at 4ºC. The separation gel buffer and stacking gel buffer are stable for at least 2 wk when stored at 4ºC.
5. APS and the upper reservoir buffer must be freshly prepared before use. The lower reservoir buffer can be used for several weeks.
6. AFLP data may be directly imported into GelCompar or BioNumerics software packages (Applied Maths) when ABI (Applied Biosystems), ALF, or MegaBace (Amersham Biotech) platforms are used. Other platforms require import from digi- tized images in a standard graphical file (e.g., TIFF).
7. SDS-PAGE analysis of whole-cell lysates is performed according to Pot et al. (21).
8. The high molecular protein bands are highly homogenous and only when a restricted protein region (20.1–36 kDa) is analyzed differentiation in genogroups is obtained.
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From: Methods in Molecular Biology, vol. 345: Diagnostic Bacteriology Protocols, Second Edition Edited by: L. O‘Connor © Humana Press Inc., Totowa, NJ