DNAs Plasmids Cloning and Transfection

Sugar Crush Detox

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1. C2GnT-I (Genebank accession no. NM 001490) and leukosialin (sialophorin, CD43; Genebank accession no. NM 003123), both in pcDNA1 vector (Invitrogen), were kindly provided by Professor Minora Fukuda (The Burnham Institute, La Jolla, CA; see also Chapter 13).

2. Oligonucleotide primers (Genset, Every, France). Forward (sense) primer for C2GnT-I: 5'-cgicgiggatccATGCTGAGGACGTTGCTGCGA-3'. BamHl site, bold letters; extranucleotides, italicized letters; the C2GnT-I sequence, capital letters, covers between start codon and codon 7. Reverse (antisense) primer for C2GnT-I: 5'-CACCATGGTGGCGaccggtGTGTTTTAATGTCTCCAA-3'. Agel site, bold letters; EGFP complementary sequence, italicized letters, covering codon -5 to codon +2 (the start codon is codon +1). The complementary sequence to C2GnT-I, capital letters, covers codon 423 to codon 428; the stop codon, codon 429, is deleted.

3. Taq polymerase, restriction enzymes, and T4-DNA ligase (Promega, Madison, WI).

4. Deionized autoclaved water (see Note 1).

5. Vectors pEGFP-Nl (Clontech, Palo Alto, CA) and pcDNA-3.1(+) (Invitrogen).

6. DNA gel extraction kit and plasmid (mini and maxi) preparation kits (Qiagen, Hilden, Germany).

7. Ultracompetent Escherichia coli Top 10F' cells.

8. Super optimal catabolite (SOC) medium: 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM of NaCl, 2.5 mM of KCl, 10 mM of MgCl2, 10 mM of MgSO4, and 20 mM of glucose (Invitrogen).

9. Luria-Bertani broth (LB): To make 1 L solution: 10 g of Bacto tryptone (Difco, Detroit, MI), 5 g of Bacto yeast extract (Difco), and 5 g of NaCl in water. Adjust the pH to 7.0. Autoclave and store at room temperature.

10. LB agar supplemented with 100 |g/mL ampicillin (stock solution 50 mg/mL in water) in plastic Petri dishes (Falcon, BD Labware, Franklin Lakes, NJ).

11. LipofectAMINE Plus reagent and neomycin (G418; Invitrogen).

2.3. Immunofluorescence

1. Falcon eight-well glass chamber slides (Becton Dickinson).

2. Antibody binding buffer: PBS containing 1% bovine serum albumin (BSA).

3. Anti-CD43 monoclonal antibody T305 (IgG) and anti-C2GnT-I poyclonal antibody (kindly provided by M. Fukuda), and anti-EGFP monoclonal antibody JL-8 (IgG, Clontech).

4. Rhodamine isothiocyanate (RITC)-labeled goat anti-mouse IgG (Sigma).

5. Fluorescence-activated cell sorter (FACSCalibur analyzer; Becton Dickinson).

6. Zeiss Axiovert 200 (Zeiss, Gottingen, Germany) fluorescence microscope. This inverted microscope is equipped with a sensitive polychrome camera (Axio-CamMRC, Zeiss) and a filter set for fluorescent probes such as EGFP and GFP variants blue fluorescent protein (BFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), as well as fluorescein isothiocyanate (FITC), rhodamine, and 4',6'-diamidino-2-phenylindole (DAPI). The MetaMorph software (v. 6.0) is used for image acquisition and image analysis. This equipment is useful for imaging the dynamic of fluorescent proteins in living cells.

2.4. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

1. 30% Acrylamide/to-acrylamide solution (29:1; Sigma), N,N,N,N'-tetramethyl-ethylenediamine (TEMED; Biorad, Hercules, CA,), and 66% (w/v) sucrose. Store at 4°C.

2. 10% Ammonium persulfate (w/v) in water (freeze immediately at -20°C in single-use 500-|L aliquots).

3. 10% Tris-HCl (w/v) sodium dodecyl sulfate (SDS) in water. Store at room temperature.

4. Separating buffer: 3 M of Tris-HCl (pH 8.8) and 1 M of stacking buffer (pH 6.8). Store at room temperature.

5. 5X Laemmli (5) SDS-sample buffer: 0.5 M of Tris-HCl (pH 6.8), 7.5% (w/v) SDS, 25 mM of EDTA, 5 M of sucrose, and 0.05% (w/v) of bromophenol blue. Store at -20°C.

6. 10X Running buffer: 0.25 M Tris-HCl, 2 M of glycine, and 1% SDS. Store at room temperature.

7. Mini Protean-II protein electrophoresis device and All Blue prestained molecular weight markers (Bio-Rad).

2.5. Western Immunoblotting

1. 1X Transfer buffer: 48 mM of Tris-HCl (no need to adjust pH), 39 mM of glycine, and 20% methanol. Semi-dry transfer cell (Bio-Rad).

2. Polyvinylidene difluorite (PVDF) membrane (Amersham, Buckinghamshire, UK) and 3MM chromatography paper (Whatman, Madison, UK).

3. Ponceau red solution (0.1% Ponceau S in 5% acetic acid; Sigma).

4. Secondary antibody: Horseradish peroxidase-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).

5. Tris-buffered saline containing Tween-20 (TBS-T): 25 mM of Tris-HCl (pH 7.4), 0.137 M of NaCl, and 0.1% Tween-20. Blocking buffer and primary/secondary dilution buffer: TBS-T containing 5% (w/v) nonfat dry milk.

6. Enhanced chemiluminescent (Supersignal) reagent (Pierce, Rockford, IL).

2.6. In Vitro Enzyme Assay for C2GnT-I

1. 5X Assay buffer: 250 mM of N-morpholino-ethanesulfonic acid (MES; Sigma), pH 7.0, containing 500 mM of N-acetylglucosamine (GlcNAc; Sigma). Set pH with 1 M of NaOH. Store at 4°C.

2. 5X Donor solution: 5 mM of uridine 5'-diphosphate (UDP)-GlcNAc (Sigma). Store in aliquots at -20°C. UDP-[6-3H]GlcNAc (36 Ci/mmol, 0.1 mCi/mL; Perkin Elmer, Boston MA).

3. 10X Acceptor solution: 10 mM of Galß1,3GalNAca-0-paranitrophenyl (Toronto Research Chemicals, Downsview ON, Canada). Dissolve in water and store at -20°C.

4. Sep-Pak columns (Sep-Pak C18 cartridges, Waters Corp, Milford, MA).

5. Vacuum dryer (Speedvac, Farmingdale, NY).

6. Liquid scintillation cocktail photon correlation spectroscopy (PCS; Amersham, Arlington Heights, IL).

7. LKB/Wallac 1214 beta-counter (Perkin Elmer).

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Appetite Antidote

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